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Biomedical subjects

Michael Keller

Publications and source records attributed to Michael Keller.

At least 19 recordsLinked to original sources
PubMed2006

MAGfect: a novel liposome formulation for MRI labelling and visualization of cells.

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.

Cells↗
PubMed2006

Condition and fate of logged forests in the Brazilian Amazon.

The long-term viability of a forest industry in the Amazon region of Brazil depends on the maintenance of adequate timber volume and growth in healthy forests. Using extensive high-resolution satellite analyses, we studied the forest damage caused by recent logging operations and the likelihood that logged forests would be cleared within 4 years after timber harvest. Across 2,030,637 km2 of the Brazilian Amazon from 1999 to 2004, at least 76% of all harvest practices resulted in high levels of canopy damage sufficient to leave forests susceptible to drought and fire. We found that 16+/-1% of selectively logged areas were deforested within 1 year of logging, with a subsequent annual deforestation rate of 5.4% for 4 years after timber harvests. Nearly all logging occurred within 25 km of main roads, and within that area, the probability of deforestation for a logged forest was up to four times greater than for unlogged forests. In combination, our results show that logging in the Brazilian Amazon is dominated by highly damaging operations, often followed rapidly by deforestation decades before forests can recover sufficiently to produce timber for a second harvest. Under the management regimes in effect at the time of our study in the Brazilian Amazon, selective logging would not be sustained.

Biomass↗
PubMed2006

Booster vaccination in the elderly: their success depends on the vaccine type applied earlier in life as well as on pre-vaccination antibody titers.

BACKGROUND: Recent retrospective studies demonstrate that elderly persons have a shortened protection period following vaccination with recall antigens. METHODS: We now analysed the effect of booster vaccination with a multivalent vaccine containing tetanus, dipththeria, pertussis and polio antigens in 252 healthy elderly persons. The magnitude of the humoral immune response was assessed by antibody measurements. RESULTS: Comparison with a small control group of 21 younger persons demonstrates that pre- and post-vaccination antibody concentrations are lower in elderly persons for all antigens except polio, for which higher pre- and similar post-vaccination antibody levels are observed. Using multiple linear regression analysis we also show that the magnitude of the humoral immune response in elderly persons greatly depends on pre-vaccination antibody concentrations in the case of tetanus, diphtheria and pertussis, but much less so in the case of polio, against which priming and preceding booster immunizations were performed with attenuated live vaccine. CONCLUSION: Regular booster vaccinations throughout life are of clinical importance to maintain the ability to respond to recall antigens in old age. Longer lasting protection and good responsiveness to boosting in spite of low antibody titres can be expected following exposure to live vaccine earlier in life.

Adult↗
PubMed2006

An empirical model of the sympatric coexistence of two strains of the endoparasitoid wasp Venturia canescens.

Recent research has demonstrated that a laboratory culture of the asexual solitary endoparasitoid wasp Venturia canescens Grav. (Hymenoptera: Ichneumonidae) contains two genetically and phenotypically distinct lines, coexisting on their host the flour moth Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). The basis to the coexistence of the two lines appears to be differences in their reproductive success under single parasitism and superparasitism. Furthermore, examination of field-derived wasps from several locations has shown that the phenotypes displayed by the laboratory colonies also co-occur in field populations. Historically, the impossibility of showing that two species do not occupy separate niches has precluded any demonstration of sympatric coexistence in the field. Here we present the results of an iterative model that uses a range of experimental life history data to predict the stable composition of a mixed population of two lines displaying the laboratory phenotypes under different rates of superparasitism. The model predicts that sympatric coexistence of the two lines is possible when the overall rate of superparasitism is between 4 and 12% or greater. These values are within the rates reported for other solitary endoparasitoid wasp species in the field, and so demonstrate that the sympatric coexistence under natural conditions of two species that display the phenotypes observed in the laboratory lines is, in principle, possible.

Animals↗
PubMed2006

Naive T cells in the elderly: are they still there?

One of the most striking changes in the primary lymphoid organs during human aging is the progressive involution of the thymus. As a consequence, the rate of naïve T cell output dramatically declines with age and the peripheral T cell pool shrinks. These changes lead to increased incidence of severe infections and decreased protective effect of vaccinations in the elderly. Little is, however, known of the composition and function of the residual naïve T cell repertoire in elderly persons. To evaluate the impact of aging on the naïve T cell pool, we investigated the quantity, phenotype, function, composition, and senescence status of CD45RA(+)CD28(+) human T cells--a phenotype generally considered as naïve cells--from both young and old healthy donors. We found a significant decrease in the number of CD45RA(+)CD28(+) T cells in the elderly, whereas the proliferative response of these cells is still unimpaired. In addition to their reduced number, CD45RA(+)CD28(+) T cells from old donors display significantly shorter telomeres and have a restricted TCR repertoire in nearly all 24 Vbeta families. These findings let us conclude that naïve T cells cannot be classified with conventional markers in old age.

Adult↗
PubMed2005

Selective logging in the Brazilian Amazon.

Amazon deforestation has been measured by remote sensing for three decades. In comparison, selective logging has been mostly invisible to satellites. We developed a large-scale, high-resolution, automated remote-sensing analysis of selective logging in the top five timber-producing states of the Brazilian Amazon. Logged areas ranged from 12,075 to 19,823 square kilometers per year (+/-14%) between 1999 and 2002, equivalent to 60 to 123% of previously reported deforestation area. Up to 1200 square kilometers per year of logging were observed on conservation lands. Each year, 27 million to 50 million cubic meters of wood were extracted, and a gross flux of approximately 0.1 billion metric tons of carbon was destined for release to the atmosphere by logging.

Brazil↗
PubMed2005

Between adaptive and innate immunity: TLR4-mediated perforin production by CD28null T-helper cells in ankylosing spondylitis.

CD3+CD4+CD28null and CD3+CD8+CD28null T cells are enriched in patients with immune-mediated diseases compared with healthy controls. This study shows that CD4+CD28null T cells express Toll-like receptors recognizing bacterial lipopolysaccharides in ankylosing spondylitis, psoriatic arthritis and rheumatoid arthritis. In ankylosing spondylitis, TLR4 (23.1 +/- 21.9%) and, to a smaller extent, TLR2 (4.1 +/- 5.8%) were expressed on CD4+CD28null T cells, whereas expression was negligible on CD4+CD28+ and CD8+ T cells. CD4+CD28null T cells produced perforin upon stimulation with lipopolysaccharide, and this effect was enhanced by autologous serum or recombinant soluble CD14. Perforin production could be prevented with blocking antibodies directed against CD14 or TLR4. Incubation of peripheral blood mononuclear cells with tumour necrosis factor alpha led to an upregulation of TLR4 and TLR2 on CD4+CD28null T cells in vitro, and treatment of patients with antibodies specifically directed against tumour necrosis factor alpha resulted in decreased expression of TLR4 and TLR2 on CD4+CD28null T cells in vivo. We describe here a new pathway for direct activation of cytotoxic CD4+ T cells by components of infectious pathogens. This finding supports the hypothesis that CD4+CD28null T cells represent an immunological link between the innate immune system and the adaptive immune system.

Adult↗
PubMed2005

Age-related differences in phenotype and function of CD4+ T cells are due to a phenotypic shift from naive to memory effector CD4+ T cells.

Based on the combined expression of CD27 and CD28, a putative model of T cell differentiation has been previously proposed. We used CD27 and CD28 expression in order to comparatively study the size, cytokine production capacity and proliferative response of CD4+ T cell sub-populations from healthy young and elderly volunteers. Elderly persons had a lower percentage of CD27+CD28+ but a higher percentage of CD27-CD28+ and CD27-CD28-CD4+ T cells than the young persons. CD27-CD28-CD4+ T cells were present, although at relatively low numbers, in the vast majority of the healthy elderly donors but were only sporadically detected in young persons. Each CD4+ T cell sub-population exhibited a distinct phenotype and cytokine production profile, which were not affected by age. When purified CD27+CD28+ were stimulated by staphylococcal enterotoxin B, they proliferated to a greater extent than CD27-CD28+ and CD27-CD28-CD4+ T cells. However, we did not observe age-related differences in proliferative response of each sub-population. We concluded that although the size of the different sub-populations differed between the young and the old group, the functional characteristics of each sub-population were the same in both age groups. This suggests that on a per cell basis there is no functional impairment of CD4 memory T cells in elderly persons. Consequently, potential differences in the function of the total CD4+ T cell population are most likely due to different composition of repertoire.

Adult↗
PubMed2005

Femtosecond degenerate four-wave mixing of cyclopropane.

Femtosecond degenerate four-wave mixing (fs-DFWM) is applied for the measurement of rotational constants of cyclopropane (C3H6). The rotational coherence method yields a very accurate B0 = 20,093.322(12) MHz and centrifugal distortion constants D(J) and D(JK). To exploit the full resolution of the fs-DFWM method, the accuracy of the optical delay measurement was increased by nearly two orders of magnitude, including elimination of effects from the refractive index of air. The fs-DFWM molecular constants are comparable in accuracy to those from high-resolution infrared spectroscopy and are only surpassed by those of dipole distortion microwave spectroscopy. In parallel, the equilibrium structure, vibrationally averaged structure parameters and rotational constants were calculated using high-level ab initio methods and large basis sets. Combining these with the results of previous calculations and the measured rotational constants yields r(e)(C-C) = 1.5034(3) A, r(e)(C-H) = 1.0775(5) A, and alpha(e)(H-C-H) = 115.09(10) degrees.

Journal Article↗
PubMed2005

CD25-expressing CD8+ T cells are potent memory cells in old age.

We have recently described an IL-2/IL-4-producing CD8+CD25+ non-regulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 alphabeta molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25- memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25- memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25- memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25- memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Aged↗
PubMed2005

Synthesis and application of integrin targeting lipopeptides in targeted gene delivery.

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl-L-alpha-phosphatidylethanolamine), mu peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target alpha(9)beta(1)-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. "Targeted" LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.

Animals↗
PubMed2005

Long-term cytomegalovirus infection leads to significant changes in the composition of the CD8+ T-cell repertoire, which may be the basis for an imbalance in the cytokine production profile in elderly persons.

In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naive (CD45RA+ CD28+), memory (CD45RA- CD28+), and effector (CD45RA+ CD28- or CD45RA- CD28-) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65(495-503)-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naive CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naive and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.

Adult↗
PubMed2004

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Apoptosis↗
PubMed2004

De-novo design of complementary (antisense) peptide mini-receptor inhibitor of interleukin 18 (IL-18).

Complementary (antisense) peptide mini-receptor inhibitors are complementary peptides designed to be receptor-surrogates that act by binding to selected surface features of biologically important proteins thereby inhibiting protein-cognate receptor interactions and subsequent biological effects. Previously, we described a complementary peptide mini-receptor inhibitor of interleukin-1beta (IL-1beta) that was designed to bind to an external surface loop (beta-bulge) of IL-1beta (Boraschi loop) clearly identified in the X-ray crystal structure of this cytokine. Here, we report the de-novo design and rational development of a complementary peptide mini-receptor inhibitor of cytokine interleukin-18 (IL-18), a protein for which there is no known X-ray crystal structure. Using sequence homology comparisons with IL-1beta, putative IL-18 surface loops are identified and used as a starting point for design, including a loop region 1 thought to be equivalent with the Boraschi loop of IL-1beta. Only loop region 1 complementary peptides are found to be promising leads as mini-receptor inhibitors of IL-18 but these are prevented from being properly successful owing to solubility problems. The application of "M-I pair mutagenesis" and inclusion of a C-terminal arginine residue are then sufficient to solve this problem and convert one lead peptide into a functional complementary peptide mini-receptor inhibitor of IL-18. This suggests that the biophysical and biological properties of complementary peptides can be improved in a rational and logical manner where appropriate, further strengthening the potential importance of complementary peptides as inhibitors of protein-protein interactions, even when X-ray crystal structural information is not readily available.

Drug Design↗
PubMed2003

Carbon in Amazon forests: unexpected seasonal fluxes and disturbance-induced losses.

The net ecosystem exchange of carbon dioxide was measured by eddy covariance methods for 3 years in two old-growth forest sites near Santarém, Brazil. Carbon was lost in the wet season and gained in the dry season, which was opposite to the seasonal cycles of both tree growth and model predictions. The 3-year average carbon loss was 1.3 (confidence interval: 0.0 to 2.0) megagrams of carbon per hectare per year. Biometric observations confirmed the net loss but imply that it is a transient effect of recent disturbance superimposed on long-term balance. Given that episodic disturbances are characteristic of old-growth forests, it is likely that carbon sequestration is lower than has been inferred from recent eddy covariance studies at undisturbed sites.

Brazil↗
PubMed2003

Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.

Biophysical Phenomena↗