New methods to approach immunotherapy of cancer – and strategies of tumours to avoid elimination

Owing to the intense effort of numerous investigators, the number of tumour antigens potentially of use for clinical immunotherapy continues to increase. At the same time, further strategies employed by tumour cells to avoid destruction by the immune system are being uncovered. A combined onslaught to target tumour cells and prevent their “escape” will be required for successful immunotherapy. Progress in this area was the subject of a meeting supported by the European Cancer Research Consortium “EUCAPS”, which was held in London in February 2000. This conference was the second of a series, the first of which was summarised previously in this journal [Pawelec G et al. (1999) Cancer Immunol Immunother 48: 214]. Received: 14 March 2000 / Accepted: 30 March 2000     

Patterns and determinants of immigrants’ sense of belonging to Canada and their source country

This study examines the relative distribution of immigrants who have: strong sense of belonging to both Canada and the source country; strong sense of belonging to Canada only; strong sense of belonging to the source country only; and weak sense of belonging to Canada and the source country. It further examines four sets of determinants of these acculturation profiles, including source-country socio-economic and cultural characteristics, immigrant entry status, post-migration experience, and demographic characteristics. Using a large national representative sample of 7,000 immigrants in Canada from over 100 countries, this study finds that the overwhelming majority of immigrants have a strong sense of belonging to Canada with or without a strong sense of belonging to their source country. Source-country attributes are as important as immigrant entry status and post-migration experience in affecting immigrants’ sense of belonging to Canada and their source country.     

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2 ± 0.2) × 10⁶ M^− 1 at 25 °C, pH 7.4 and I = 0.15 M. Dependence of K_B on pH and on the monomer-dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer-dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0 ± 1.5) × 10⁵ M^− 1 at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.     

Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K_spm-β-LG = 3.2(±0.6) × 10⁴ M⁻¹, K_spmd-β-LG = 1.8(±0.5) × 10⁴ M⁻¹, K_BE-333-β-LG = 5.8(±0.3) × 10⁴ M⁻¹ and K_{BE-3333-β-LG} = 6.2(±0.05) × 10⁴ M⁻¹. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.     

Contribution of ΔpH and ΔE to Photosynthesis of Chlamydomonas Reinhardtii

The contribution of two components (pH and E) of the proton motive force to photosynthesis of C. reinhardtii was studied. Valinomycin, a photophosphorylation uncoupler, decreased significantly the fast phase (related mainly to the membrane electric potential) of millisecond delayed light emission (ms-DLE) of C. reinhardtii. Nigericin, another photophosphorylation uncoupler, decreased the slow phase (related mainly to the proton gradient) and partly also the fast phase of ms-DLE. Both valinomycin and nigericin decreased the net ATP content and photosynthetic rate of C. reinhardtii, but the inhibition by nigericin was stronger than that by valinomycin. Hence both components of the proton motive force contribute to photosynthesis and although the contribution of pH is larger than that of E, the latter is not negligible in photosynthesis of C. reinhardtii.     

Time and energy constraints and the evolution of sexual size dimorphism — to eat or to mate?

Summary We present an empirical test of the Ghiselin—Reiss small-male hypothesis for the evolution of sexual size dimorphism (SSD). In mating systems dominated by scramble competition, where male reproductive success is a function of encounter rate with females, small males may be favoured when food is limiting because they require lower absolute amounts of food. Given a trade-off between time and energy devoted to foraging and to mate acquisition, small males should be able to devote more time to the latter. If at the same time larger females are favoured, this mechanism will contribute to the evolution of SSD and may be the major determinant of the female-biased SSDs that characterize most animal taxa. We tested this hypothesis using the water strider,Aquarius remigis (Heteroptera: Gerridae), a scramble competitor which mates many times over a prolonged mating season and which shows female-biased SSD. Laboratory experiments demonstrated that foraging success and giving up times (GUTs) are lower for males than for females during the reproductive season and that male water striders flexibly alter their time budgets under conditions of energy limitation. Controlled feeding experiments showed that male and female longevity, female fecundity and male mating success are positively related to food availability. As predicted, male body size is negatively correlated with several indices of male fitness (longevity, number of mating attempts and mating success), while female body size is positively correlated with longevity. These results are consistent with the hypothesis that scramble competition for mates favours small males in this species and provides empirical support for the Ghiselin—Reiss small-male hypothesis.     

Use of Fat-Fed Rats to Study the Metabolic and Vascular Sequelae of Obesity and ��-Adrenergic Antagonism

Obesity-associated cardiovascular disease exerts profound human and monetary costs, creating a mounting need for cost-effective and relevant in vivo models of the complex metabolic and vascular interrelationships of obesity. Obesity is associated with endothelial dysfunction and inflammation. Free fatty acids (FFA), generated partly through β-adrenergic receptor-mediated lipolysis, may impair endothelium-dependent vasodilation (EDV) by proinflammatory mechanisms. β-Adrenergic antagonists protect against cardiovascular events by mechanisms not fully defined. We hypothesized that β antagonists may exert beneficial effects, in part, by inhibiting lipolysis and reducing FFA. Further, we sought to evaluate the fat-fed rat as an in vivo model of obesity-induced inflammation and EDV. Control and fat-fed rats were given vehicle or β antagonist for 28 d. Serum FFA were measured to determine the association to serum IL6, TNFα, and C-reactive protein and to femoral artery EDV. Compared with controls, fat-fed rats weighed more and had higher FFA, triglyceride, leptin, and insulin levels. Unexpectedly, in control and fat-fed rats, β antagonism increased FFA, yet inflammatory cytokines were reduced and EDV was preserved. Therefore, reduction of FFA is unlikely to be the mechanism by which β antagonists protect the endothelium. These results reflect the need for validation of ex vivo models of obesity-induced inflammation and endothelial dysfunction, concurrent with careful control of dietary fat composition and treatment duration.Abbreviations: CRP, C-reactive protein; EDV, endothelium-dependent vasodilation; FFA, free fatty acids; FTI, flow–time integral; L-NAME, N_ω-nitro-L-arginine methyl ester; MAP, mean arterial pressure; PKA, protein kinase AThe prevalence of overweight and obese adults in the United States has increased by almost 20% over the last 3 decades.³⁶ Similar upward trends have been observed in persons between 6 and 19 y of age.³⁷ The obesity epidemic extracts a monetary cost of more than $92 billion on medical care alone⁵⁶ and a profound human price in the form of increased disease³⁵ and higher death rates.⁵⁰Obese adults have a higher risk of morbidity and mortality due to cardiovascular disease.⁵⁷ In health, endothelial cells that line the luminal surface of blood vessels release mediators that facilitate the appropriate regulation of multiple processes, including vascular permeability, inflammation and cell adhesion, coagulation, maintenance of intercellular matrix, lipid metabolism, and vascular reactivity.^25,42 Dysregulation of these processes favors inflammation, coagulation, and vasoconstriction. Not surprisingly, endothelial dysfunction as measured by impairment of endothelium-dependent vasodilation (EDV) is an early and reliable predictor of cardiovascular events in humans.^43,47Obese persons have increased serum free fatty acids (FFA).¹ Obesity^14,53 and FFA¹³ are associated with increased circulating inflammatory markers, specifically IL6, TNFα, and C-reactive protein (CRP). In addition, both obesity³² and FFA^8,46 are associated with impaired EDV, and FFA exert direct adverse inflammatory effects on the endothelium.¹⁸Partly in response to stimulation of β-adrenergic receptors, FFA are the principle moiety secreted from adipocytes. β-Adrenergic antagonist drugs reduce morbidity and mortality in patients with coronary artery disease^9,26 and affect both the myocardium^2,38 and vasculature.^5,51,55 The mechanisms by which β-adrenergic antagonists exert protection remain unclear, but reduced generation of FFA might play a role.The first aim of the present study was to determine whether β antagonism lowers serum FFA in fat-fed rats and whether the magnitude and direction of change in FFA is correlated with circulating inflammatory markers and EDV. The β₁- and β₃-receptor subtypes predominantly mediate lipolysis in rodent adipose;^12,30 however, to minimize the potential for compensatory upregulation of unopposed receptors in this study, the β₁β₂ antagonist propranolol was combined with the β₃ antagonist SR59230A (Sigma-Aldrich, St Louis, MO) to exert antagonism at all 3 receptor subtypes. To test whether the high-fat dietary treatment was associated with a metabolic milieu consistent with obesity, serum triglycerides, leptin, glucose, and insulin concentrations were measured.An inexpensive, valid, and physiologically relevant in vivo system would be valuable for studying the pandemic of obesity³¹ and related endothelial dysfunction.¹⁷ We are unaware of studies of whether long-term high-fat feeding affects in vivo EDV in the rat, although a study validating the use of high-resolution ultrasonography to measure in vivo flow-mediated vasodilation in normal rats was published recently.¹⁷ The second aim of our study was to investigate the fat-fed rat as a model of human diet-induced endothelial dysfunction. To retain the complex metabolic-vascular interplay that occurs in the intact organism, we used in vivo measures of EDV to assess the integrated physiologic response. In humans, the dilator response of peripheral vessels is associated with coronary EDV response.⁴⁸ We studied the rat femoral artery, with the aim of demonstrating changes in vasodilator responses in this easily isolated peripheral vascular bed.     

Binding of Sulfamethazine to β-cyclodextrin and Methyl-β-cyclodextrin

β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) complexes with sulfamethazine (SMT) were prepared and characterized by different experimental techniques, and the effects of βCD and MβCD on drug solubility were assessed via phase-solubility analysis. The phase-solubility diagram for the drug showed an increase in water solubility, with the following affinity constants calculated: 40.4 ± 0.4 (pH 2.0) and 29.4 ± 0.4 (pH 8.0) M⁻¹ with βCD and 56 ± 1 (water), 39 ± 3 (pH 2.0) and 39 ± 5 (pH 8.0) M⁻¹ with MβCD. According to ¹H NMR and 2D NMR spectroscopy, the complexation mode involved the aromatic ring of SMT included in the MβCD cavity. The complexes obtained in solid state by freeze drying were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and thermal analysis. The amorphous complexes obtained in this study may be useful in the preparation of pharmaceutical dosage forms of SMT.     

Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β(2)-microglobulin (β(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β(2)m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β(2)m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β(2)m fibrils, and that both monomeric β(2)m and fibrillar β(2)m are cytotoxic to these cells. β(2)m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β(2)m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β(2)m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β(2)m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.     

Kinds of glycosidase and properties of β-N-acetylglucosaminidase bound to mycelia of streptomyces sp.

An extract liberated from mycelia of the L-13 strain of Streptomyces by 0.2 M phosphate buffer, pH 6.5, contained β-N-acetylglucosaminidase, β-N-acetylgalactosaminidase and a little α-glucosidase. On chromatofocusin, the isoelectric point of β-N-acetylglucosaminidase was around pH 8.1. The enzyme prepared thus was homogeneous, and had both β-N-acetylglucosaminidase and β-N-acetlygalactosaminidase activities. The β-N-acetylglucosaminidase was most active at pH 6.0 and stable between pH 4 to 8. The K_m value for p-nitrophenyl β-N-acetylglucosaminide was 0.25 mM. N-Acetylglucosaminolactone was the most potent inhibitor tested.     

Synthesis of the tri- and tetra-saccharides related to the fine structures of lichenan and cereal β-d-glucans

Syntheses, based on silver trifluoromethanesulfonate-promoted Koenigs-Knorr type condensations, are described of the d-glucotrioses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp and β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, and the d-Glucotetraoses, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-d-Glcp, β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-d-Glcp, and β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glcp, corresponding to the tri- and tetra-saccharide units in the linear chains of (1→4)- and (1→3)-linked β-d-glucopyranosyl residues of lichenan, and of oat and barley β-d-glucans.     

Safety of the production process of SURFACEN® to inactivate and remove virus

SURFACEN^® is a biological product produced from pig lungs. Since these animals can be potential sources of microbial pathogens such as viruses, the manufacturing process of this product should guarantee safety from health hazards. The SURFACEN^® production procedure is capable of effective viral clearance (inactivation/removal) by involving two stages of organic solvent extraction followed by acetone precipitation and heat treatment. In this study, we evaluated the clearance capacity of these four stages for a wide range of viruses by performing spiking experiments. Residual contamination was assessed using a Tissue Culture Infectious Dose assay (log₁₀ TCID₅₀). The validation study demonstrated that, for all viruses tested, the TCID₅₀ titers were reduced by more than 2 log₁₀ in each stage. Total log reduction values achieved were between ≥17.82 log₁₀ and ≥27.93 log₁₀, depending on the virus physical properties, titer, and the number of processing stages applied. Results indicated that the production procedure of SURFACEN^® can inactivate or remove contaminant viruses from the raw material.     

Sensitivity of ortho- and paramyxovirus replication to human interferon α

Replication of the influenza virus strains Influenza Ao/WSN (H0N1), fowl plague (Hav1N1) and B-Lee/40 (ATCC) and the paramyxovirus, New Castle disease virus (Victoria) are highly sensitive to human interfereon type in Madin Darby bovine kidney cells. Pretreatment of cells with human interferon type resulted in protection of the cells against viral cytopathic effect. The inhibition of the orthomyxovirus strains used in this study and New Castle disease virus replication is mediated by an inhibition of viral protein synthesis. Residual WSN virus particles released from interferon treated cells showed the same structural protein pattern as virus particles isolated from control cells. Glycosylation of the viral structural components appeared to be unaffected by interferon.     

Cleavage of BLM and Sensitivity of Bloom’s Syndrome Cells to Hydroxurea

Patients with Bloom’s syndrome (BS) show a strong genetic instability and a predisposition to all types of cancer. Here, we report that the Bloom’s syndrome protein (BLM) is cleaved in response to hydroxyurea (HU)- or UVC-induced apoptosis. The appearance and solubility of BLM proteolytic products differed according to whether proteolysis occurred in response to HU or UVC. One BS cell line homozygous for a null mutation in BLM was resistant to both UVC- and HU-induced apoptosis, while another one expressing a mutated BLM protein was resistant to HU-induced apoptosis but displayed normal sensitivity to UVC. Thus, UVC and HU appear to induce apoptosis through distinct pathways.

Key Words:

Bloom’s syndrome, Apoptosis, Hydroxyurea, Ultraviolet C radiation, RecQ helicase     

Structural Characterization of LNA and α-L-LNA Hybridized to RNA

Abstract

LNA and α-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we present a short overview of the structural results we have obtained for LNA:RNA and α-L-LNA:RNA hybrids. Specifically, we have shown that LNA acts as an A-type mimic, while α-L-LNA acts as a B-type mimic when built into oligonucleotides.     

Synthesis and Biological Activities of New Carbaacyclonucleosides and 1′-Oxaacyclonucleosides Related to Clitocine

Abstract

We describe the synthesis of two series of acyclonucleosides: carbaacyclonucleosides and 1′-oxaacyclonucleosides which possess the same aglycone as clitocine 3 which is a natural nucleoside exhibiting interesting biological properties. These compounds have been obtained by condensation of 4-aminobutanol or 3-silyloxypropoxyamine with 4,6-dichloro-5-nitropyrimidine. Structural modifications have been made on the heterocyclic base and the side chain to enhance their potential activity.     

Discovery and characterization of a large number of diagnostic markers to discriminate Oncorhynchus mykiss and O. clarkii

Hybridization of cutthroat trout and steelhead/rainbow trout is ubiquitous where they are sympatric, either naturally or owing to introductions. The ability to detect hybridization and introgression between the two species would be greatly improved by the development of more diagnostic markers validated across the two species' many phylogenetic lineages. Here, we describe 81 novel genetic markers and associated assays for discriminating the genomes of these sister species. These diagnostic nucleotide polymorphisms were discovered by sequencing of rainbow trout expressed sequence tags (ESTs) in a diverse panel of both cutthroat trout and steelhead/rainbow trout. The resulting markers were validated in a large number of lineages of both species, including all extant subspecies of cutthroat trout and most of the lineages of rainbow trout that are found in natural sympatry with cutthroat trout or used in stocking practices. Most of these markers (79%) distinguish genomic regions for all lineages of the two species, but a small number do not reliably diagnose coastal, westslope and/or other subspecies of cutthroat trout. Surveys of natural populations and hatchery strains of trout and steelhead found rare occurrences of the alternative allele, which may be due to either previous introgression or shared polymorphism. The availability of a large number of genetic markers for distinguishing genomic regions originating in these sister species will allow the detection of both recent and more distant hybridization events, facilitate the study of the evolutionary dynamics of hybridization and provide a powerful set of tools for the conservation and management of both species.     

Remote sensing of biodiversity: what to measure and monitor from space to species?

Global biodiversity monitoring systems through remote sensing can support consistent assessment, monitoring, modelling and reporting on biodiversity which are key activities intended for sustainable management. This work presents an overview of biodiversity monitoring components, i.e. biodiversity levels, essential biodiversity variables, biodiversity indicators, scale, biodiversity inventory, biodiversity models, habitat, ecosystem services, vegetation health and biogeochemical heterogeneity and discusses what remote sensing through Earth Observations has contributed to the study of biodiversity. The technological advancements in remote sensing have enabled information-rich data on biodiversity. Remote sensing data are making a strong contribution in providing unique information relevant to various biodiversity research and conservation applications. The extensive use of Earth observation data are not yet realized in biodiversity assessment, monitoring and conservation. The development of direct remote sensing approaches and the techniques for quantifying biodiversity at the community to species level is likely to be a great challenge for comprehensive earth observation-based monitoring strategy.