Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Characterization of a new class of DNA delivery complexes formed by the local anesthetic bupivacaine

Bupivacaine, a local anesthetic and cationic amphiphile, forms stable liposomal-like structures upon direct mixing with plasmid DNA in aqueous solutions. These structures are on the order of 50-70 nm as determined by scanning electron microscopy, and are homogeneous populations as analyzed by density gradient centrifugation. The DNA within these structures is protected from nuclease degradation and UV-induced damage in vitro. Bupivacaine:DNA complexes have a negative zeta potential (surface charge), homogeneous nature, and an ability to rapidly assemble in aqueous solutions. Bupivacaine:DNA complexes, as well as similar complexes of DNA with other local anesthetics, have the potential to be a novel class of DNA delivery agents for gene therapy and DNA vaccines.     

Phylogeny of South African Gnaphalieae (Asteraceae) based on two noncoding chloroplast sequences

The Gnaphalieae are a group of sunflowers that have their greatest diversity in South America, Southern Africa, and Australia. The objective of this study was to reconstruct a phylogeny of the South African Gnaphalieae using sequence data from two noncoding chloroplast DNA sequences, the trnL intron and trnL/trnF intergenic spacer. Included in this investigation are the genera of the Gnaphalieae from the African basal groups, members of the subtribes Cassiniinae, Gnaphaliinae, and Relhaniinae, and African representatives from the large Old World genus Helichrysum. Results indicate that two Gnaphaloid genera, Printzia and Callilepis, should be excluded from the Gnaphalieae. In most trees the Relhaniinae s.s. (sensu stricto) and some of the basal taxa comprise a clade that is sister to the remainder of the tribe Gnaphalieae. The Relhaniinae, which are restricted to Africa, are not a monophyletic group as presently circumscribed, nor are the South African members of Helichrysum, the Cassiniinae and Gnaphaliinae. There is general agreement between our molecular analysis and that of morphology, particularly in the terminal branches of the trees.     

Cellulosomes-structure and ultrastructure

The cellulosome is a macromolecular machine, whose components interact in a synergistic manner to catalyze the efficient degradation of cellulose. The cellulosome complex is composed of numerous kinds of cellulases and related enzyme subunits, which are assembled into the complex by virtue of a unique type of scaffolding subunit (scaffoldin). Each of the cellulosomal subunits consists of a multiple set of modules, two classes of which (dockerin domains on the enzymes and cohesin domains on scaffoldin) govern the incorporation of the enzymatic subunits into the cellulosome complex. Another scaffoldin module-the cellulose-binding domain-is responsible for binding to the substrate. Some cellulosomes appear to be tethered to the cell envelope via similarly intricate, multiple-domain anchoring proteins. The assemblage is organized into dynamic polycellulosomal organelles, which adorn the cell surface. The cellulosome dictates both the binding of the cell to the substrate and its extracellular decomposition to soluble sugars, which are then taken up and assimilated by normal cellular processes.     

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.     

Significance of Relative Position of Cellulases in Designer Cellulosomes for Optimized Cellulolysis

Degradation of cellulose is of major interest in the quest for alternative sources of renewable energy, for its positive effects on environment and ecology, and for use in advanced biotechnological applications. Due to its microcrystalline organization, celluose is extremely difficult to degrade, although numerous microbes have evolved that produce the appropriate enzymes. The most efficient known natural cellulolytic system is produced by anaerobic bacteria, such as C. thermocellum, that possess a multi-enzymatic complex termed the cellulosome. Our laboratory has devised and developed the designer cellulosome concept, which consists of chimaeric scaffoldins for controlled incorporation of recombinant polysaccharide-degrading enzymes. Recently, we reported the creation of a combinatorial library of four cellulosomal modules comprising a basic chimaeric scaffoldin, i.e., a CBM and 3 divergent cohesin modules. Here, we employed selected members of this library to determine whether the position of defined cellulolytic enzymes is important for optimized degradation of a microcrystalline cellulosic substrate. For this purpose, 10 chimaeric scaffoldins were used for incorporation of three recombinant Thermobifida fusca enzymes: the processive endoglucanase Cel9A, endoglucanase Cel5A and exoglucanase Cel48A. In addition, we examined whether the characteristic properties of the T. fusca enzymes as designer cellulosome components are unique to this bacterium by replacing them with parallel enzymes from Clostridium thermocellum. The results support the contention that for a given set of cellulosomal enzymes, their relative position within a scaffoldin can be critical for optimal degradation of microcrystaline cellulosic substrates.     

Projection of Young-Old and Old-Old with Functional Disability: Does Accounting for the Changing Educational Composition of the Elderly Population Make a Difference?

This study compares projections, up to year 2040, of young-old (aged 60-79) and old-old (aged 80+) with functional disability in Singapore with and without accounting for the changing educational composition of the Singaporean elderly. Two multi-state population models, with and without accounting for educational composition respectively, were developed, parameterized with age-gender-(education)-specific transition probabilities (between active, functional disability and death states) estimated from two waves (2009 and 2011) of a nationally representative survey of community-dwelling Singaporeans aged ≥60 years (N=4,990). Probabilistic sensitivity analysis with the bootstrap method was used to obtain the 95% confidence interval of the transition probabilities. Not accounting for educational composition overestimated the young-old with functional disability by 65 percent and underestimated the old-old by 20 percent in 2040. Accounting for educational composition, the proportion of old-old with functional disability increased from 40.8 percent in 2000 to 64.4 percent by 2040; not accounting for educational composition, the proportion in 2040 was 49.4 percent. Since the health profiles, and hence care needs, of the old-old differ from those of the young-old, health care service utilization and expenditure and the demand for formal and informal caregiving will be affected, impacting health and long-term care policy.     

Attachment,Symptom Severity,and Depression in Medically Unexplained Musculoskeletal Pain and Osteoarthritis: A Cross-Sectional Study

Background

Attachment insecurity relates to the onset and course of chronic pain via dysfunctional reactions to pain. However, few studies have investigated the proportion of insecure attachment styles in different pain conditions, and results regarding associations between attachment, pain severity, and disability in chronic pain are inconsistent. This study aims to clarify the relationships between insecure attachment and occurrence or severity of chronic pain with and without clearly defined organic cause. To detect potential differences in the importance of global and romantic attachment representations, we included both concepts in our study.

Methods

85 patients with medically unexplained musculoskeletal pain (UMP) and 89 patients with joint pain from osteoarthritis (OA) completed self-report measures of global and romantic attachment, pain intensity, physical functioning, and depression.

Results

Patients reporting global insecure attachment representations were more likely to suffer from medically unexplained musculoskeletal pain (OR 3.4), compared to securely attached patients. Romantic attachment did not differ between pain conditions. Pain intensity was associated with romantic attachment anxiety, and this relationship was more pronounced in the OA group compared to the UMP group. Both global and romantic attachment anxiety predicted depression, accounting for 15% and 17% of the variance, respectively. Disability was independent from attachment patterns.

Conclusions

Our results indicate that global insecure attachment is associated with the experience of medically unexplained musculoskeletal pain, but not with osteoarthritis. In contrast, insecure attachment patterns seem to be linked to pain intensity and pain-related depression in unexplained musculoskeletal pain and in osteoarthritis. These findings suggest that relationship-informed focused treatment strategies may alleviate pain severity and psychological distress in chronic pain independent of underlying pathology.     

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased K_d value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R⁴⁹ that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding.