Major histocompatibility complex genes partly explain early survival in house sparrows.

Environmental factors and genetic incompatibilities between parents have been suggested as important determinants for embryonic mortality and survival. The genetic set-up of the immune system, specifically the highly polymorphic major histocompatibility complex (MHC) may also influence individual resistance to infections. MHC proteins are important for an appropriate adaptive immune response and enable T-cells to separate 'self' from 'non-self'. Here we investigate the importance of MHC functional diversity for early development in birds, more specifically, if offspring survival and body mass or size depends on number of different functional MHC alleles, specific functional MHC alleles or similarity of MHC alleles in the parents. Unhatched eggs are common in clutches of many bird species. In house sparrows (Passer domesticus), embryo and nestling mortality can exceed 50%. To control for environmental factors, our study was carried out on an aviary population. We found that one specific functional MHC allele was associated with reduced nestling survival, which was additionally supported by lower body mass and a smaller tarsus when nestlings have been 6 days old. Another allele was positively associated with tarsus length at a later nestling stage (nestlings 12 days old). These results indicate that MHC alleles might influence pathogen resistance or susceptibility.

Evolution of antigenic diversity in the tick-transmitted bacterium Borrelia afzelii: a role for host specialization?

Antigenic diversity in pathogenic microbes can be a result of at least three different processes: diversifying selection by acquired immunity, host-pathogen coevolution and/or host specialization. Here, we investigate whether host specialization drives diversity at ospC (which encodes an immunodominant surface protein) in the tick-transmitted bacterium Borrelia afzelii. We determined prevalence and infection intensity of ospC strains in naturally infected wild mammals (rodents and shrews) by 454 amplicon sequencing in combination with qPCR. Neither prevalence nor infection intensity of specific ospC strains varied in a species-specific manner (i.e. there were no significant ospC × host species interactions). Rankings of ospC prevalences were strongly positively correlated across host species. Rankings of ospC infection intensities were correlated more weakly, but only in one case significantly < 1. ospC prevalences in the studied mammals were similar to those in ticks sampled at the study site, indicating that we did not miss any mammal species that are important hosts for specific ospC strains. Based on this, we conclude that there is at best limited host specialization in B. afzelii and that other processes are likely the main drivers of ospC diversity.

The evolution of highly variable immunity genes across a passerine bird radiation.

To survive, individuals must be able to recognize and eliminate pathogens. The genes of the major histocompatibility complex (MHC) play an essential role in this process in vertebrates as their diversity affects the repertoire of pathogens that can be recognized by the immune system. Emerging evidence suggests that birds within the parvorder Passerida possess an exceptionally high number of MHC genes. However, this has yet to be directly investigated using a consistent framework, and the question of how this MHC diversity has evolved has not been addressed. We used next-generation sequencing to investigate how MHC class I gene copy number and sequence diversity varies across the Passerida radiation using twelve species chosen to represent the phylogenetic range of this group. Additionally, we performed phylogenetic analyses on this data to identify, for the first time, the evolutionary model that best describes how MHC class I gene diversity has evolved within Passerida. We found evidence of multiple MHC class I genes in every family tested, with an extremely broad range in gene copy number across Passerida. There was a strong phylogenetic signal in MHC gene copy number and diversity, and these traits appear to have evolved through a process of Brownian motion in the species studied, that is following the pattern of genetic drift or fluctuating selection, as opposed to towards a single optimal value or through evolutionary 'bursts'. By characterizing MHC class I gene diversity across Passerida in a systematic framework, this study provides a first step towards understanding this huge variation.

Sexual antagonism in the pistil varies among populations of a hermaphroditic mixed-mating plant.

Sexual conflicts and their evolutionary outcomes may be influenced by population-specific features such as mating system and ecological context; however, very few studies have investigated the link between sexual conflict and mating system. The self-compatible, mixed-mating hermaphrodite Collinsia heterophylla (Plantaginaceae) is thought to exhibit a sexual conflict over timing of stigma receptivity. This conflict involves (i) delayed stigma receptivity, which intensifies pollen competition, and (ii) early fertilization forced by pollen, which reduces seed set. We investigated the potential for the conflict to occur under field conditions and performed glasshouse crosses within eight populations to assess its consistency across populations. Flowers were visited, and produced seeds after pollination, at all developmental stages, suggesting that the conflict can be of significance under natural conditions. In the glasshouse, early pollination imposed costs in all populations. Overall, the timing of first seed set was most strongly affected by the maternal parent, denoting stronger female than male ability to influence the onset of stigma receptivity. Crosses also revealed a negative relationship between donor- and recipient-related onset of receptivity within individuals, a novel result hinting at trade-offs in sex allocation or a history of antagonistic selection. Neither timing of stigma receptivity, timing of first seed set, nor pollen competitive ability covaried with population outcrossing rate. In conclusion, these results indicate that sexually antagonistic selection may be present in varying degrees in different populations of C. heterophylla, but this variation does not appear to be directly related to mating system variation.

Transcriptional analysis of the pheromone gland of the turnip moth, Agrotis segetum (Noctuidae), reveals candidate genes involved in pheromone production.

Moths generally rely on pheromone communication for mate finding. The pheromone components of most moths are produced by a common pathway of fatty-acid biosynthesis coupled with species-specific modifications of the final products. Some genes involved in moth pheromone production have previously been described, whereas others remain to be characterized and thus the molecular mechanisms accounting for the production of species-specific blends are far from understood. The turnip moth, Agrotis segetum, has a multicomponent pheromone, consisting of at least four components derived from palmitic and stearic acid. Different populations produce and respond to different pheromone blends, which makes this species an excellent model for research on genes and molecular mechanisms involved in moth pheromone production. For this purpose, we performed an expressed sequence tag (EST) analysis of two cDNA libraries, one representing the female pheromone gland and the other representing the remainder of the insect body. Among 2285 ESTs analysed altogether, we identified a unigene set of 707 putative gene representatives. The comparative distribution of those in the two libraries showed the transcriptomes of the tissues to be clearly different. One third of the gene representatives were exclusively found in the pheromone gland. From sequence homology to public database information we assigned putative functional roles for a majority of the unigenes and then compared functional profiles of the two tissues. In the set of ESTs more abundant in the pheromone gland library, we found homologues of an acyl-CoA Delta11-desaturase, a G-protein subunit, a chemosensory protein as well as a juvenile hormone binding protein.

Analysis of carbohydrates using liquid chromatography--surface plasmon resonance immunosensing systems.

An immunosensing system based on surface plasmon resonance (SPR) was used for on-line detection and characterization of carbohydrate molecules separated by high-performance liquid chromatography. These analytes, with or without serum, were continuously separated and analyzed in the combined liquid chromatography-surface plasmon resonance (LC-SPR) system. By using weak and readily reversible monoclonal antibodies, the SPR system allowed specific on-line monitoring of the substances. To increase the specificity of the immunosensor, nonrelevant antibodies were used as reference in a serial flow cell. The sensitivity of the LC-SPR system was dependent on molecular weight of the carbohydrate, affinity of binding, and design of the sensor.

Continuous weak-affinity immunosensing.

A multitude of weak biological interactions, either working alone or in concert, occur frequently throughout biological systems. We have used this natural feature of readily reversible interactions as the basis for continuous immunosensing. In a model system, a set of weak monoclonal antibodies directed towards a carbohydrate epitope was studied with the aid of surface plasmon resonance. Because the system requires no regeneration, it can be used as a truly on-line immunosensing device. This principle should have wide application in all areas where there is a need for the continuous evaluation of a molecule.

New approach to steroid separation based on a low affinity IgM antibody.

IgM antibodies are often of low affinity (dissociation constant (Kd) > 10(-5) M) and therefore they are usually neglected as tools in, e.g., immunoassays. Previous studies have shown that low affinity biological interactions can be studied and exploited in affinity chromatography, biosensor technology and capillary electrophoresis. In this study we have demonstrated that IgM can be a useful ligand for analytical separation of antigens in weak affinity chromatography (WAC). A low affinity human monoclonal IgM antibody, directed at digoxin, was produced in a hybridoma cell culture, purified to homogeneity and immobilized onto an HPLC support. The IgM HPLC column displayed specific weak affinity retention in the 0.01-0.1 mM range as evaluated with digoxin and ouabain. The specificity was not affected when samples of ouabain in a crude environment of diluted serum were separated on the IgM column. These findings suggest an approach in immunoadsorbent technology where biomolecules can be analyzed and separated with weak affinity chromatography using IgM as a general affinity ligand.

Studies of interactions with weak affinities and low-molecular-weight compounds using surface plasmon resonance technology.

Interactions between the immobilized weak-affinity monoclonal IgG antibody 39.5, which is specific for the glucose-alpha 1,4-glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 degrees C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 degrees C) to 1.0 mM (40 degrees C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (< 1000 Da), a careful design of control surfaces is demanded to exclude artefactual results.

Detection and characterization of weak affinity antibody antigen recognition with biomolecular interaction analysis.

In biological systems, weak-affinity interactions (association constant, Ka, of less than approximately 10(4) M-1) between biomolecules are common and essential to the integrity of such units. However, studies of weak biological interactions are difficult due to the scarcity of analytical methods available for the bioscientist. In this communication, we report on the use of biosensors based on surface plasmon resonance to detect and characterize weak affinity antibody-antigen interactions. Monoclonal antibodies towards carbohydrate antigens were immobilized on sensor surfaces and were used to detect weak binding of the carbohydrate tetraglucose of dissociation constant, Kd, in the millimolar range. Sensorgrams were received in the form of square pulses where the kinetic rate constants were difficult to assess due to the rapid association and dissociation of the antigen to/from the immobilized antibody.

In vivo absorbed dose measurements with mini-TLDs--parameters affecting the reliability.

Mini-TLDs have been proposed and widely used for in vivo measurements of absorbed doses in radionuclide therapies. The present investigation reports in detail on the signal dependence on different parameters and the accuracy of this method. Rodshaped Teflon-imbedded CaSO4:Dy or LiF thermoluminescent dosimeters (TLDs) with dimensions 0.2 x 0.4 x 5 mm3 were prepared from TLD-discs. To remove paraffin from the mini-TLDs after cutting in a microtome the TLDs were Xylene-treated, which does not affect the sensitivity. Irradiated mini-TLDs are sensitive to illumination. Fading effects in darkness were examined after 60Co-irradiation at temperatures 4, 22 and 37 degrees C. For CaSO4:Dy mini-TLDs fading in air is small. The observed signal loss after implanting CaSO4:Dy mini-TLDs in gel and muscle tissue is the same at constant temperature and is increasing with the temperature. For LiF mini-TLDs the effect of signal loss in gel was smaller than for CaSO4:Dy dosimeters. For 60Co external irradiation supralinearity already starts between 0.5 and 1 Gy for both kinds of dosimeter material. There is a strong pH dependence of the signals from the mini-TLDs. For CaSO4:Dy dosimeters the loss of sensitivity in gel is smaller at higher pHs. For LiF dosimeters the loss of sensitivity is smallest for neutral pH. We conclude that using mini-TLDs for in vivo dosimetry requires careful handling and proper calibration for accuracy in the measurements. Without such calibration errors exceeding 65% for CaSO4:Dy and 40% for LiF may easily occur.

Parameters to consider for measurements of absorbed doses in vivo with mini-thermoluminescent dosimeters.

BACKGROUND: For systemic radiation therapy, i.e., radioimmunotherapy, there is a demand for direct methods of measuring the absorbed dose in vivo. One such method is the use of mini-thermoluminescent dosimeters (TLDs). This paper reports an investigation of the sensitivity of tissue implanted mini-TLDs (calcium sulfate:Dy, 0.2 x 0.4 x 5.0 mm). METHODS: After being irradiated with cobalt-60, the mini-TLDs were left for as long as 9 days in air, gel, and muscle tissue. RESULTS: There was an extensive signal loss, which increased with time, except in air. After 9 days in gel or muscle tissue at room temperature, the signal was decreased to one third of its original value. The dosimeters needed to be kept in constant darkness. There was a strong pH dependence, with a loss of sensitivity of 63% at a pH below 5, which got smaller at higher pH values and reached 10% at pH = 10. CONCLUSIONS: When using mini-TLDs in vivo, one must calibrate the dosimeters in similar milieus, unless the position of the dosimeters in tissue after implantation can be monitored for temperature, pH, and liquid flow.

Considerations on the calibration of small thermoluminescent dosimeters used for measurement of beta particle absorbed doses in liquid environments.

An investigation has been carried out on the factors which affect the absolute calibration of thermoluminescent dosimeters (TLDs) used in beta particle absorbed dose evaluations. Four effects on light output (LO) were considered: decay of detector sensitivity with time, finite TLD volume, dose linearity, and energy dependence. Most important of these was the decay of LO with time in culture medium, muscle tissue, and gels. This permanent loss of sensitivity was as large as an order of magnitude over a 21-day interval for the nominally 20-microns-thick disc-shaped CaSO4(Dy) TLDs in gel. Associated leaching of the dosimeter crystals out of the Teflon matrix was observed using scanning electron microscopy. Large channels leading from the outside environment into the TLDs were identified using SEM images. A possibility of batch dependence of fading was indicated. The second most important effect was the apparent reduction of light output due to finite size and increased specific gravity of the dosimeter (volume effect). We estimated this term by calculations as 10% in standard "mini" rods for beta particles from 90Y, but nearly a factor of 3 for 131I beta particles in the same geometry. No significant nonlinearity of the log (light output) with log (absorbed dose) over the range 0.05-20.00 Gy was discovered. Energy dependence of the LO was found to be not detectable, within measurement errors, over the range of 0.60-6.0 MeV mean energy electrons. With careful understanding of these effects, calibration via gel phantom would appear to be an acceptable strategy for mini TLDs used in beta absorbed dose evaluations in media.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron microscopy and computed microtomography studies of in vivo implanted mini-TL dosimeters.

The need for direct methods of measuring the absorbed dose in vivo increases for systemic radiation therapy, and in more sophisticated methodologies developed for radioimmunotherapy. One method suggested is the use of mini-thermoluminescent dosimeters (TLD). Recent reports indicate a marked loss of signal when the dosimeters are used in vivo. We investigated the exterior surface of the dosimeters with scanning electron microscopy and the interior dosimeter volume with computed microtomography. The results show that the dosimeters initially have crystals uniformly embedded in the teflon matrix, with some of them directly exposed to the environment. After incubation in gel, holes appear in the dosimeter matrix where the crystals should have been. The computed microtomographic images show that crystals remain in the interior of the matrix, producing the remaining signal. We conclude that these dosimeters should be very carefully handled, and for practical use of mini-TLDs in vivo the dosimeters should be calibrated in equivalent milieus. An alternative solution to the problem of decreased TL efficiency, would be to coat the dosimeters with a thin layer, of Teflon, or other suitable material.