The future direction of cholesterol-lowering therapy

PURPOSE OF REVIEW: Observational studies suggest a continuous positive relationship between vascular risk and cholesterol without any lower threshold level. We review recent and future clinical trials addressing the question of optimal treatment goals for cholesterol reduction and how these relate to present guidelines. With increasing focus on greater cholesterol reduction, new approaches to lipid-lowering therapy are being developed; we discuss some of these agents including the new statin, rosuvastatin and novel cholesterol transport inhibitors such as ezetimibe. RECENT FINDINGS: The Heart Protection Study demonstrated that LDL cholesterol reduction to levels as low as 1.7 mmol/l was associated with significant clinical benefit in a wide range of high-risk individuals, irrespective of baseline cholesterol levels, with no apparent threshold level for LDL cholesterol with respect to cardiovascular risk. The Heart Protection Study also demonstrated that the benefits of LDL cholesterol reduction extend into peripheral vascular disease and cerebrovascular disease prevention and suggest that the most recent National Cholesterol Education Program Adult Treatment Panel III guidelines, with LDL cholesterol targets of 2.6 mmol/l, may result in undertreatment of a large number of patients. Various large end-point trials, including Treating to New Targets and Study of Effectiveness of Additional Reductions in Cholesterol and Homocysteine will attempt to further address the issue of optimal LDL cholesterol reduction. New therapies are being developed to meet the challenge of more intensive cholesterol lowering. Rosuvastatin is a potent, hydrophilic enantiomeric statin producing reductions in LDL cholesterol of 40-69% over its dose range of 5-80 mg. Ezetimibe is a selective cholesterol absorption inhibitor, with a site of action at the intestinal epithelium. Optimum reductions in LDL cholesterol of up to 25 and 60% reduction in chylomicron cholesterol content are seen with a 10-mg dose. SUMMARY: Evidence is accumulating supporting the safety and benefits of aggressive cholesterol reduction, with no apparent threshold for LDL cholesterol. New therapies will aid in achieving lower cholesterol levels and the use of combination therapies targeting different aspects of cholesterol metabolism may produce additional benefits. Outcome studies are awaited to further address these issues.     

Factors Affecting Phage D29 Infection: A Tool to Investigate Different Growth States of Mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non-pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay.     

Overshooting dynamics in a model adaptive radiation

The history of life is punctuated by repeated periods of unusually rapid evolutionary diversification called adaptive radiation. The dynamics of diversity during a radiation reflect an overshooting pattern with an initial phase of exponential-like increase followed by a slower decline. Much attention has been paid to the factors that drive the increase phase, but far less is known about the causes of the decline phase. Decreases in diversity are rarely associated with climatic changes or catastrophic events, suggesting that they may be an intrinsic consequence of diversification. We experimentally identify the factors responsible for losses in diversity during the later stages of the model adaptive radiation of the bacterium Pseudomonas fluorescens. Proximately, diversity declines because of the loss of biofilm-forming niche specialist morphotypes. We show that this loss occurs despite the presence of strong divergent selection late in the radiation and is associated with continued adaptation of resident niche specialists to both the biotic and abiotic environments. These results suggest that losses of diversity in the latter stages of an adaptive radiation may be a general consequence of diversification through competition and lends support to the idea that the conditions favouring the emergence of diversity are different from those that ensure its long-term maintenance.     

Whole-molecule antibody engineering: generation of a high-affinity anti-IL-6 antibody with extended pharmacokinetics

The differentiation of therapeutic monoclonal antibodies in an increasingly competitive landscape requires optimization of clinical efficacy combined with increased patient convenience. We describe here the generation of MEDI5117, a human anti-interleukin (IL)-6 antibody generated by variable domain engineering, to achieve subpicomolar affinity for IL-6, combined with Fc (fragment crystallizable) engineering to enhance pharmacokinetic half-life. MEDI5117 was shown to be highly potent in disease-relevant cellular assays. The pharmacokinetics of MEDI5117 were evaluated and compared to those of its progenitor, CAT6001, in a single-dose study in cynomolgus monkeys. The antibodies were administered, either subcutaneously or intravenously, as a single dose of 5 mg/kg. The half-life of MEDI5117 was extended by approximately 3-fold, and clearance was reduced by approximately 4-fold when compared to CAT6001. MEDI5117 therefore represents a potential ‘next-generation’ antibody; future studies are planned to determine the potential for affinity-driven efficacy and/or less frequent administration.     

Multivariate Phenotypic Divergence Due to the Fixation of Beneficial Mutations in Experimentally Evolved Lineages of a Filamentous Fungus

The potential for evolutionary change is limited by the availability of genetic variation. Mutations are the ultimate source of new alleles, yet there have been few experimental investigations of the role of novel mutations in multivariate phenotypic evolution. Here, we evaluated the degree of multivariate phenotypic divergence observed in a long-term evolution experiment whereby replicate lineages of the filamentous fungus Aspergillus nidulans were derived from a single genotype and allowed to fix novel (beneficial) mutations while maintained at two different population sizes. We asked three fundamental questions regarding phenotypic divergence following approximately 800 generations of adaptation: (1) whether divergence was limited by mutational supply, (2) whether divergence proceeded in relatively many (few) multivariate directions, and (3) to what degree phenotypic divergence scaled with changes in fitness (i.e. adaptation). We found no evidence that mutational supply limited phenotypic divergence. Divergence also occurred in all possible phenotypic directions, implying that pleiotropy was either weak or sufficiently variable among new mutations so as not to constrain the direction of multivariate evolution. The degree of total phenotypic divergence from the common ancestor was positively correlated with the extent of adaptation. These results are discussed in the context of the evolution of complex phenotypes through the input of adaptive mutations.     

Genotype × environment interaction analysis of North American shrub willow yield trials confirms superior performance of triploid hybrids

Development of dedicated bioenergy crop production systems will require accurate yield estimates, which will be important for determining many of the associated environmental and economic impacts of their production. Shrub willow (Salix spp) is being promoted in areas of the USA and Canada due to its adaption to cool climates and wide genetic diversity available for breeding improvement. Willow breeding in North America is in an early stage, and selection of elite genotypes for commercialization will require testing across broad geographic regions to gain an understanding of how shrub willow interacts with the environment. We analyzed a dataset of first‐rotation shrub willow yields of 16 genotypes across 10 trial environments in the USA and Canada for genotype‐by‐environment interactions using the additive main effects and multiplicative interactions (AMMI) model. Mean genotype yields ranged from 5.22 to 8.58 oven‐dry Mg ha^?1 yr^?1. Analysis of the main effect of genotype showed that one round of breeding improved yields by as much as 20% over check cultivars and that triploid hybrids, most notably Salix viminalis × S. miyabeana, exhibited superior yields. We also found important variability in genotypic response to environments, which suggests specific adaptability could be exploited among 16 genotypes for yield gains. Strong positive correlations were found between environment main effects and AMMI parameters and growing environment temperatures. These findings demonstrate yield improvements are possible in one generation and will be important for developing cultivar recommendations and for future breeding efforts.     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.     

The Phenotypic Characterization of the Human Renal Mononuclear Phagocytes Reveal a Co-Ordinated Response to Injury

Mammalian tissues contain networks of mononuclear phagocytes (MPh) that sense injury and orchestrate the response to it. In mice, this is affected by distinct populations of dendritic cells (DC), monocytes and macrophages and recent studies suggest the same is true for human skin and intestine but little is known about the kidney. Here we describe the analysis of MPh populations in five human kidneys and show they are highly heterogeneous and contain discrete populations of DC, monocytes and macrophages. These include: plasmacytoid DC (CD303⁺) and both types of conventional DC—cDC1 (CD141⁺ cells) and CD2 (CD1c⁺ cells); classical, non-classical and intermediate monocytes; and macrophages including a novel population of CD141⁺ macrophages clearly distinguishable from cDC1 cells. The relative size of the MPh populations differed between kidneys: the pDC population was bi-modally distributed being less than 2% of DC in two kidneys without severe injury and over 35% in the remaining three with low grade injury in the absence of morphological evidence of inflammation. There were profound differences in the other MPh populations in kidneys with high and low numbers of pDC. Thus, cDC1 cells were abundant (55 and 52.3%) when pDC were sparse and sparse (12.8–12.5%) when pDC were abundant, whereas the proportions of cDC2 cells and classical monocytes increased slightly in pDC high kidneys. We conclude that MPh are highly heterogeneous in human kidneys and that pDC infiltration indicative of low-grade injury does not occur in isolation but is part of a co-ordinated response affecting all renal DC, monocyte and macrophage populations.