CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope

Cytotoxic CD8+ T cells (CTLs) play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF). We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D) and a majority encoding an asparagine (Q9VF/5N). We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.     

Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase

Apolipoprotein A5 (APOA5) is associated with differences in triglyceride levels and familial combined hyperlipidemia. In genetically engineered mice, apoAV plasma levels are inversely correlated with plasma triglycerides. To elucidate the mechanism by which apoAV influences plasma triglycerides, metabolic studies and in vitro assays resembling physiological conditions were performed. In human APOA5 transgenic mice (hAPOA5tr), catabolism of chylomicrons and very low density lipoprotein (VLDL) was accelerated due to a faster plasma hydrolysis of triglycerides by lipoprotein lipase (LPL). Hepatic VLDL and intestinal chylomicron production were not affected. The functional interplay between apoAV and LPL was further investigated by cross-breeding a human LPL transgene with the apoa5 knock-out and the hAPOA5tr to an lpl-deficient background. Increased LPL activity completely normalized hypertriglyceridemia of apoa5-deficient mice; however, overexpression of human apoAV modulated triglyceride levels only slightly when LPL was reduced. To reflect the physiological situation in which LPL is bound to cell surface proteoglycans, we examined hydrolysis in the presence or absence of proteoglycans. Without proteoglycans, apoAV derived either from triglyceride-rich lipoproteins, hAPOA5tr high density lipoprotein, or a recombinant source did not alter the LPL hydrolysis rate. In the presence of proteoglycans, however, apoAV led to a significant and dose-dependent increase in LPL-mediated hydrolysis of VLDL triglycerides. These results were confirmed in cell culture using a proteoglycan-deficient cell line. A direct interaction between LPL and apoAV was found by ligand blotting. It is proposed, that apoAV reduces triglyceride levels by guiding VLDL and chylomicrons to proteoglycan-bound LPL for lipolysis.     

Variation von PCB-Gemischen in Eiern und V?geln des Wattenmeeres

Zusammenfassung Bei Flußseeschwalbe und Austernfischer wurde der Einfluß von Standort und Jahr auf die Zusammensetzung der PCB-Gemische im Ei untersucht. Die Gesamtkonzentration, der Metabolisierungsgrad und die Ringstabilitätszahlen des Gemisches von 45 bestimmten PCB-Kongeneren wurden erfaßt. In den Mündungsbereichen von Elbe, Weser und in der Inneren Deutschen Bucht traten nicht nur die höchsten PCB-Konzentrationen auf, sondern auch die am weitesten metabolisierten Gemische. 1987–1989 zeigte sich im Metabolisierungsgrad bei beiden Vogelarten ein linearer Anstieg. Flußseeschwalbeneier enthielten zwar deutlich höhere PCB-Konzentrationen als Austernfischereier, doch war das Gemisch geringer metabolisiert. Deutliche Artunterschiede traten auch in den Ringstabilitätszahlen auf. Zur Beurteilung der PCB-Belastung sollte deshalb in einem Monitoring-Programm der Metabolisationsgrad auch mitberücksichtigt werden.

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Variation of PCB mixtures in eggs of birds of the Wadden Sea

Summary The influence of location and year on the composition of PCB mixtures found in eggs was examined in the two wadden sea bird species Common Tern (Sterna hirundo) and Oystercatcher (Haematopus ostralegus). By means of 45 PCB congeners the total concentration, the degree of metabolism and the ring stability values were analysed. The highest PCB concentrations as well as the highest degree of metabolism were found in the estuaries of the rivers Elbe and Weser and in the Deutsche Bucht. The degree of metabolism of both species increased linearly from 1987 to 1989. The total PCB concentration in the eggs of Common Terns were clearly higher than those found in the eggs of Oystercatchers but the mixture was less metabolised. Furthermore, there are significant differences of the ring stability values between the two species. To assess the PCB incrimination the degree of metabolism should be considered as well in a monitoring program.

     

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.     

An siRNA-based system for differential regulation of ectopic gene expression constructs

We describe an siRNA (short interfering RNA)-based approach for temporary and reversible regulation of engineered expression constructs in cells. Control of cloned genes can be achieved by cotransfection of unique siRNAs, complementary to artificial target sequences, which are integral parts of an expression vector. Application of this method allows simultaneous or mutual-differential regulation of two or more gene constructs within the same cell, reducing unwanted side effects. This method provides several advantages over promoter regulatory systems employing chemical compounds.     

Delineation of marker chromosomes by reverse chromosome painting using only a small number of DOP-PCR amplified microdissected chromosomes

A new procedure for determining the chromosomal origin of marker chromosomes has been carried out. The origin of marker chromosomes that were unidentifiable by standard banding techniques could be verified by reverse chromosome painting. This technique includes microdissection, followed by in vitro DNA amplification and fluorescence in situ hybridization (FISH). A number of marker chromosomes prepared from unbanded and from GTG-banded lymphocyte chromosomes were collected with microneedles and transferred to a collection drop. The chromosomal material was amplified by a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The resulting PCR products were labelled by nick-translation with biotin-11-dUTP and used as probes for FISH. They were hybridized onto normal metaphase spreads in order to determine the precise regional chromosomal origin of the markers. Following this approach, we tested 2–14 marker chromosomes in order to determine how many are necessary for reverse chromosome painting. As few as two marker chromosomes provided sufficient material to paint the appropriate chromosome of origin, regardless of whether the marker contained heterochromatic or mainly euchromatic material. With this method, it was possible to identify two marker chromosomes of a healthy proband [karyotype: 48,XY, +mar₁,+mar₂] and an aberrant Y chromosome of a mentally retarded boy [karyotype: 46,X, der(Y)].     

Carbon Dioxide in Boreal Surface Waters: A Comparison of Lakes and Streams

The quantity of carbon dioxide (CO₂) emissions from inland waters into the atmosphere varies, depending on spatial and temporal variations in the partial pressure of CO₂ (pCO₂) in waters. Using 22,664 water samples from 851 boreal lakes and 64 boreal streams, taken from different water depths and during different months we found large spatial and temporal variations in pCO₂, ranging from below atmospheric equilibrium to values greater than 20,000???atm with a median value of 1048???atm for lakes (n?=?11,538 samples) and 1176???atm for streams (n?=?11,126). During the spring water mixing period in April/May, distributions of pCO₂ were not significantly different between stream and lake ecosystems (P?>?0.05), suggesting that pCO₂ in spring is determined by processes that are common to lakes and streams. During other seasons of the year, however, pCO₂ differed significantly between lake and stream ecosystems (P?<?0.0001). The variable that best explained the differences in seasonal pCO₂ variations between lakes and streams was the temperature difference between bottom and surface waters. Even small temperature differences resulted in a decline of pCO₂ in lake surface waters. Minimum pCO₂ values in lake surface waters were reached in July. Towards autumn pCO₂ strongly increased again in lake surface waters reaching values close to the ones found in stream surface waters. Although pCO₂ strongly increased in the upper water column towards autumn, pCO₂ in lake bottom waters still exceeded the pCO₂ in surface waters of lakes and streams. We conclude that throughout the year CO₂ is concentrated in bottom waters of boreal lakes, although these lakes are typically shallow with short water retention times. Highly varying amounts of this CO₂ reaches surface waters and evades to the atmosphere. Our findings have important implications for up-scaling CO₂ fluxes from single lake and stream measurements to regional and global annual fluxes.