Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.     

The dual AngII/AVP receptor gene N119S/C163R variant exhibits sodium-induced dysfunction and cosegregates with salt-sensitive hypertension in the Dahl salt-sensitive hypertensive rat model

BACKGROUND: Essential hypertension is a prevalent complex polygenic disease and a major risk factor for cardiovascular disease, the leading cause of death in developed countries. Because of its complex and multifactorial nature, its genetic determinants still remain largely unknown. The Dahl salt-sensitive hypertensive rat model exhibits impaired sodium handling, which is hypothesized to play a key role in the pathophysiology of polygenic hypertension. Thus, genes associated with renal regulation of salt and water balance are a priori likely candidates for a causative role in hypertension pathogenesis. The functional properties and renal-specific expression of the recently characterized AngII/AVP receptor suggest a putative modulator role in tubular sodium and fluid reabsorption. Based on these observations, we investigated the potential involvement of the AngII/AVP receptor in salt-sensitive hypertension. MATERIALS AND METHODS:We performed cosegregation analysis of the AngII/AVP receptor locus with salt-sensitive hypertension in an F2 (Dahl S X Dahl salt-resistant [R]) hybrid male cohort characterized for blood pressure by radiotelemetry after 8 weeks of high salt challenge. Further molecular analysis was done to identify putative AngII/AVP receptor molecular variants that could account for the AngII/ AVP receptor involvement in salt-sensitive hypertension pathogenesis. RESULTS:The AngII/AVP receptor was mapped to rat chromosome 1, 1.7 cM centromeric to the D1Rat188 marker by radiation hybrid mapping analysis. Quantitative trait locus (QTL) analysis detected a highly significant linkage of the AngII/AVP receptor locus with high blood pressure (LRS = 13.8, p= 0.0002). Molecular characterization of the Dahl S and Dahl R AngII/AVP receptor cDNAs revealed two amino acid substitutions in the Dahl S AngII/AVP receptor (N119S, C163R) when compared to the Dahl R AngII/AVP receptor. These mutations are associated with an increased receptor affinity for both ligands (AVP and AngII) and an enhanced G(s)-coupling by the receptor resulting in increased activation of adenylate cyclase with concomitant increase in cAMP production. CONCLUSIONS: The observed molecular dysfunction in the Dahl S AngII/AVP receptor is consistent with increased tubular sodium and fluid reabsorption observed in Dahl S rats. Interestingly, the AngII/AVPr locus is within the narrowed chromosome 1 QTL region for blood pressure detected in different rat intercross linkage analyses. Altogether, the data strongly suggest that the AngII/AVP receptor is a hypertension susceptibility gene in the Dahl S rat model, as well as raises the hypothesis that it too underlies the chromosome 1 blood pressure QTL identified in other hypertension rat models.     

Chromosome 21: a small land of fascinating disorders with unknown pathophysiology

In the year 2000 we celebrated the sequencing of the entire long arm of human chromosome 21. This achievement now provides unprecedented opportunities to understand the molecular pathophysiology of trisomy 21, elucidate the mechanisms of all monogenic disorders of chromosome 21, and discover genes and functional sequence variations that predispose to common complex disorders. All of that requires the functional analysis of gene products in model organisms, and the determination of the sequence variation of this chromosome.     

Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.     

Genome-wide linkage analysis of longitudinal phenotypes using σ<Superscript>2</Superscript><Subscript>A</Subscript> random effects (SSARs) fitted by Gibbs sampling

The study of change in intermediate phenotypes over time is important in genetics. In this paper we explore a new approach to phenotype definition in the genetic analysis of longitudinal phenotypes. We utilized data from the longitudinal Framingham Heart Study Family Cohort to investigate the familial aggregation and evidence for linkage to change in systolic blood pressure (SBP) over time. We used Gibbs sampling to derive sigma-squared-A-random-effects (SSARs) for the longitudinal phenotype, and then used these as a new phenotype in subsequent genome-wide linkage analyses. Additive genetic effects (sigma2A.time) were estimated to account for approximately 9.2% of the variance in the rate of change of SBP with age, while additive genetic effects (sigma2A) were estimated to account for approximately 43.9% of the variance in SBP at the mean age. The linkage results suggested that one or more major loci regulating change in SBP over time may localize to chromosomes 2, 3, 4, 6, 10, 11, 17, and 19. The results also suggested that one or more major loci regulating level of SBP may localize to chromosomes 3, 8, and 14. Our results support a genetic component to both SBP and change in SBP with age, and are consistent with a complex, multifactorial susceptibility to the development of hypertension. The use of SSARs derived from quantitative traits as input to a conventional linkage analysis appears to be valuable in the linkage analysis of genetically complex traits. We have now demonstrated in this paper the use of SSARs in the context of longitudinal family data.     

The effects of Eucalyptus terpenes on hepatic cytochrome P450 CYP4A, peroxisomal Acyl CoA oxidase (AOX) and peroxisome proliferator activated receptor alpha (PPARalpha) in the common brush tail possum (Trichosurus vulpecula)

Eucalyptus leaves contain a high proportion of essential oils comprising of a complex mixture of monoterpenes such as 1,8-cineole, alpha-pinene, d-limonene and p-cymene. In this study, hepatic levels of microsomal lauric acid hydroxylase and peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activities were examined in livers of possums given an artificial diet consisting of the above monoterpenes for 10 days. These values were compared with those of possums fed a control diet containing only fruits and cereals. Peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activity was significantly higher in livers of treated possums relative to that of control possums (2.96+/-0.93 vs. 0.98+/-0.88 nmol/mg protein per min, P<0.01) (mean+/-S.D., n=4). A small increase in microsomal lauric acid hydroxylase activity was observed in the treated possum in comparison with the control group (4.40+/-0.85 vs. 3.60+/-0.48 nmol/mg protein per min) (mean+/-S.D., n=4). A higher NAD/ NADP ratio was observed in treated possums as compared with control possums (4.73+/-0.65 vs. 3.51+/-0.64 nmol/mg protein per min, P<0.05) (mean+/-S.D., n=4). No other statistically significant differences in pyridine nucleotide contents were found between control and treated possums. Northern blot analysis of mRNA from rat, human, terpene treated and control possum livers, using the corresponding koala cDNA probes, detected a more intense acyl CoA oxidase (AOX) mRNA band in livers of terpene fed possums. Negligible differences in the intensity of CYP4A and PPARalpha mRNA bands were observed between the two groups. These data suggest that Eucalyptus terpenes elevate hepatic AOX expression in possums.     

Synthesis of a novel family of diterpenes and their evaluation as anti-inflammatory agents

The synthesis and biological evaluation of a new family of diterpenes, represented by structures 2 and 3, is presented. These compounds constitute isomeric analogues of acanthoic acid (1) and were examined as potent anti-inflammatory agents. Among them, methyl ester 12 exhibited a low non-specific cytotoxicity, inhibited TNF-alpha synthesis and displayed good specificity in suppressing cytokine expression.     

Food spectrum of the deepwater squid Moroteuthis ingens (Cephalopoda: Onychoteuthidae) in New Zealand waters

Prey species of the deepwater squid Moroteuthis ingens were examined for 37 large specimens captured in New Zealand waters. Caecum contents were predominantly less than 80% full and covered a range of digestion stages. The diet consisted of fish (at least seven species, of which four were myctophids) and squid. The most abundant prey was the myctophid Lampanyctodes hectoris, which was represented by 1323 otoliths from 22 caeca. The second most abundant prey was viperfish (Chauliodus sloani) and/or dragonfish (Stomias boa), represented by 537 otoliths from 17 caeca. Individual squid appeared to ingest surprisingly large numbers of fish (up to 100) during a single feeding period and could achieve feeding rates greater than 10% of their body weight per day. While some males appeared to ingest larger numbers of L. hectoris, females targeted significantly larger individuals of L. hectoris thereby ingesting a greater biomass of fish. Received: 31 July 1997 / Accepted: 15 January 1998     

Effects of NaHCO3 loading on acid-base balance, lactate concentration, and performance in racing greyhounds

This investigation examined the effects ofNaHCO₃ loading on lactateconcentration ([La]), acid-base balance, and performance for a 603.5-m sprint task. Ten greyhounds completed aNaHCO₃ (300 mg/kg body weight) andcontrol trial in a crossover design. Results are expressed as means ± SE. Presprint differences (P < 0.05) were found for NaHCO₃ vs.control, respectively, for blood pH (7.47 ± 0.01 vs. 7.42 ± 0.01), HCO₃ (28.4 ± 0.4 vs. 23.5 ± 0.3 meq/l), and base excess (5.0 ± 0.3 vs. 0.2 ± 0.3 meq/l). Peak blood [La] increased(P < 0.05) inNaHCO₃ vs. control (20.4 ± 1.6 vs. 16.9 ± 1.3 mM, respectively). Relative to control,NaHCO₃ produced a greater(P < 0.05) reduction in blood baseexcess (18.5 ± 1.4 vs. 14.1 ± 0.8 meq/l) andHCO₃ (17.4 ± 1.2 vs.12.8 ± 0.7 meq/l) from presprint to postexercise. Postexercise peak muscle H⁺concentration ([H⁺])was higher (P < 0.05) inNaHCO₃ vs. control (158.8 ± 8.8 vs. 137.0 ± 5.3 nM, respectively). Muscle[H⁺] recoveryhalf-time (7.2 ± 1.6 vs. 11.3 ± 1.6 min) and time to predosevalues (22.2 ± 2.4 vs. 32.9 ± 4.0 min) were reduced(P < 0.05) inNaHCO₃ vs. control, respectively.No differences were found in blood[H⁺] or blood[La] recovery curves or performance times.NaHCO₃ increased postexerciseblood [La] but did not reduce the muscle or blood acid-basedisturbance associated with a 603.5-m sprint or significantly affectperformance.