Assessment of protein biomarkers for preoperative differential diagnosis between benign and malignant ovarian tumors.

OBJECTIVE: To estimate the diagnostic value of tumor and immune related proteins in the discrimination between benign and malignant adnexal masses, and between different subgroups of tumors. METHODS: In this exploratory diagnostic study, 254 patients with an adnexal mass scheduled for surgery were consecutively enrolled at the University Hospitals Leuven (128 benign, 42 borderline, 22 stage I, 55 stage II-IV, and 7 secondary metastatic tumors). The quantification of 33 serum proteins was done preoperatively, using multiplex high throughput immunoassays (Luminex) and electrochemiluminescence immuno-assay (ECLIA). We calculated univariable areas under the Receiver Operating Characteristic Curves (AUCs). To discriminate malignant from benign tumors, multivariable ridge logistic regression with backward elimination was performed, using bootstrapping to validate the resulting AUCs. RESULTS: CA125 had the highest univariable AUC to discriminate malignant from benign tumors (0.85, 95% confidence interval 0.79-0.89). Combining CA125 with CA72.4 and HE4 increased the AUC to 0.87. For benign vs borderline tumors, CA125 had the highest univariable AUC (0.74). For borderline vs stage I malignancy, no proteins were promising. For stage I vs II-IV malignancy, CA125, HE4, CA72.4, CA15.3 and LAP had univariable AUCs ≥0.80. CONCLUSIONS: The results confirm the dominant role of CA125 for identifying malignancy, and suggest that other markers (HE4, CA72.4, CA15.3 and LAP) may help to distinguish between stage I and stage II-IV malignancies. However, further research is needed, also to investigate the added value over clinical and ultrasound predictors of malignancy, focusing on the differentiation between subtypes of malignancy.

Severe fetal and neonatal hyperthyroidism years after surgical treatment of maternal Graves' disease.

BACKGROUND: Fetal/neonatal hyperthyroidism is a well-known complication of maternal Graves' disease with high concentrations of TSH-receptor antibodies (TRAb). Few data are available on the management of fetal hyperthyroidism in surgically treated Graves' disease. METHODS: Clinical, ultrasound and biochemical data are reported in a fetus/neonate whose mother underwent a thyroidectomy > 10 years before and whose sibling was thin and hyperthyroid at birth. RESULTS: Maternal TRAb were persistently > 40 U/l; unequivocal signs of fetal hyperthyroidism were identified at 29 weeks gestational age (GA). The fetus was treated through maternal antithyroid drug (ATD) administration; the dose was reduced gradually once fetal tachycardia and valve dysfunction disappeared and normal T4 was confirmed by fetal blood sampling. Maternal euthyroidism was maintained. The neonate showed normal growth for GA and T4 concentration at birth but severe hyperthyroidism relapsed from day 13 until day 58. TSH remained strongly suppressed throughout the pre- and postnatal course. CONCLUSIONS: Prenatal ATD in a taper-off regime allowed normal T4 and growth in a hyperthyroid fetus from a thyroidectomised Graves' mother. Fetal TSH cannot be used to adjust the ATD dose. Prenatal ATD appears to postpone the onset but does not affect the severity or duration of the neonatal hyperthyroid flare.

Measurable Serum Estradiol and Estrone in Women 36-56 Years During Adjuvant Treatment With Aromatase Inhibitors for a Hormone Receptor-Positive Breast Cancer. Case Studies and Cross-sectional Study Using an Ultra-sensitive LC-MS/MS-Method.

BACKGROUND AND PURPOSE: Ovarian function recovery (OFR) during adjuvant use of an aromatase inhibitor (AI) negatively impacts breast cancer outcome. We measured serum FSH and estrogen levels in consecutive AI-users with an uncertain menopausal status during follow-up and report associated risk factors of OFR METHODS: A retrospective cross sectional observational monocentric study including breast cancer patients in follow-up using an adjuvant AI, age 36 to 56 years, with at least one serum estradiol (E2) and estrone (E1) measurement between 2013 and 2020. Estrogens were quantified using a sensitive liquid chromatography-tandem mass spectrometry method (LC-MS/MS). Women on LHRH agonist were included while those with a bilateral oophorectomy or ovarian irradiation were not. We aimed to identify risk factors of OFR considering age, body mass index (BMI), previous chemotherapy and duration of AI use. Univariable analysis was used to evaluate risk factors of OFR. RESULTS: E2/E1 levels were assessed in 207 patients with a median age of 50 years (range 36-56). 17 of 159 on AI (10.7%) and 3 of 48 on AI + LHRH (6.3%) had OFR. Seven out of 17 patients (41,2%) with OFR in the AI only group and 2 out of 3 patients (66,7%) in the AI+LHRH agonist group were in amenorrhea. Age <50 y and adjuvant chemotherapy were statistically significantly different between the OFR group and the group with postmenopausal estrogen levels. CONCLUSION: Breast cancer patients aged 36 to56 years need to be monitored closely during adjuvant treatment with aromatase inhibitors: to confirm menopausal status, to evaluate compliance and to ensure ovarian activity remains adequately suppressed. Estrone might be a better marker then estradiol to detect ovarian reactivation.

Hydrocarbon signatures of egg maternity, caste membership and reproductive status in the common wasp.

In most ants, bees, and wasps, the workers are capable of challenging the reproductive monopoly of the queen by laying unfertilized, male eggs. An important mechanism that can resolve this conflict is policing, whereby the queen or workers prevent successful worker reproduction by selectively eating worker-laid eggs or by attacking egg-laying workers. Egg policing by workers has been shown to occur in several social wasp species, but the information used by worker wasps to discriminate between queen-laid and worker-laid eggs has never been investigated. Our aim, therefore, was to investigate if hydrocarbons might be used in egg policing by workers in the common wasp, Vespula vulgaris, where worker policing previously has been shown to be effective. Our results show that 51 different hydrocarbons are present on the surface of newly-laid eggs, and that there are pronounced quantitative differences in the hydrocarbon profiles of queen-laid and worker-laid eggs, with longer-chained alkenes and methylated alkanes (C(28)-C(31)) in particular being more abundant on the surface of queen-laid eggs. We further show that the hydrocarbon profiles on the surface of queen-laid and worker-laid eggs resemble those found on the mother queen's and workers' cuticles. Interestingly, longer-chained methylated alkanes also were more abundant on the cuticle of both mother queens and reproductive workers, suggesting that these compounds are linked to fertility, as has also been found to be the case in several ant species.

Levels of clonal mixing in the black bean aphid Aphis fabae, a facultative ant mutualist.

Aphids are a worldwide pest and an important model in ecology and evolution. Little is known, however, about the genetic structure of their colonies at a microgeographic level. For example, it remains largely unknown whether most species form monoclonal or polyclonal colonies. Here, we present the first detailed study on levels of clonal mixing in a nonsocial facultative ant mutualist, the black bean aphid Aphis fabae. In contrast to the earlier suggestion that colonies of this species are generally monoclonal, we found that across two subspecies of the black bean aphid, A. fabae cirsiiacanthoidis and A. fabae fabae, 32% and 67% of the aphid colonies were in fact polyclonal, consisting of a mix of up to four different clones, which resulted in an overall average relatedness within colonies of 0.90 and 0.79 in the two subspecies. Data further show that the average relatedness in A. f. cirsiiacanthoidis remained relatively constant throughout the season, which means that clonal erosion due to clonal selection more or less balanced with the influx of new clones from elsewhere. Nevertheless, relatedness tended to decrease over the lifetime of a given colony, implying that clonal mixing primarily resulted from the joining of pre-existing colonies as opposed to via simultaneous host colonisation by several foundresses. Widespread clonal mixing is argued to affect the ecology and evolution of the aphids in various important ways, for example with respect to the costs and benefits of group living, the evolution of dispersal and the interaction with predators as well as with the ant mutualists.

Colony stage and not facultative policing explains pattern of worker reproduction in the Saxon wasp.

Inclusive fitness theory predicts that in colonies of social Hymenoptera headed by a multiple-mated queen, workers should benefit from policing eggs laid by other workers. Foster & Ratnieks provided evidence that in the vespine wasp Dolichovespula saxonica, workers police other workers' eggs only in colonies headed by a multiple-mated queen, but not in those headed by a single-mated one. This conclusion, however, was based on a relatively small sample size, and the original study did not control for possible confounding variables such as the seasonal colony progression of the nests. Our aim, therefore, was to reinvestigate whether or not facultative worker policing occurs in D. saxonica. Remarkably, our data show that in the studied Danish population, there was no correlation between worker-worker relatedness and the percentage of worker-derived males. In addition, we show that variability in cuticular hydrocarbon profiles among the workers did not significantly correlate with relatedness and that workers therefore probably did not have sufficient information on queen mating frequency from the workers' cuticular hydrocarbon profiles. Hence, there was no evidence that workers facultatively policed other workers' eggs in response to queen mating frequency. Nevertheless, our data do show that the seasonal progression of the nest and the location in which the males were reared both explain the patterns of worker reproduction found. Overall, our results suggest that the earlier evidence for facultative worker policing in D. saxonica may have been caused by accidental correlations with certain confounding variables, or, alternatively, that there are large interpopulation differences in the expression of worker policing.

Detection of secondary metastatic breast cancer by measurement of plasma CA 15.3.

BACKGROUND: Most current guidelines do not recommend the serial analysis of tumour marker CA 15.3 in the follow-up of asymptomatic patients treated for early breast cancer (EBC). These guidelines are based on small-scale studies carried out in an era with more limited treatment options than today. In our large academic centre, serial measurements of CA 15.3 are used routinely in the follow-up of EBC, whereas imaging for distant metastases is only carried out on indication. PATIENTS AND METHODS: In this retrospective single-centre study, patients were included if they were treated for EBC between 1 January 2000 and 1 January 2018, diagnosed with secondary metastatic disease at least 6 months after initial surgery and had CA 15.3 available at the time of diagnosis of metastases. The primary objective was to evaluate the proportion of patients in whom metastatic disease was discovered by an increasing CA 15.3. Information on the method of metastases detection, CA 15.3 evolution and survival was collected after approval of the ethics committee. RESULTS: At the moment of diagnosis of metastases, 451 of 730 included patients (62%) had CA 15.3 levels above the upper limit of normal (>30 kU/l). In 269 patients (37%), an increasing CA 15.3 was the first sign that led to the diagnosis of metastases. This was most frequent in luminal A-like tumours (48%) and in liver (45%) and bone (41%) localisation of metastases. By contrast, reported symptoms triggered the diagnosis of metastatic disease in 48% of the patients. Median overall survival was significantly longer when the relapse was discovered by CA 15.3 elevation versus those discovered by another trigger (abnormal clinical examination or history, abnormal laboratory tests or an incidental finding) (35 versus 22 months; P = 0.0027). CONCLUSION: When CA 15.3 is systematically used in the follow-up of EBC patients, the diagnosis of metastatic disease is made in 37% by a CA 15.3 increase.

Non-invasive diagnosis of endometriosis based on a combined analysis of six plasma biomarkers.

BACKGROUND: Lack of a non-invasive diagnostic test contributes to the long delay between onset of symptoms and diagnosis of endometriosis. The aim of this study was to evaluate the combined performance of six potential plasma biomarkers in the diagnosis of endometriosis. METHODS: This case-control study was conducted in 294 infertile women, consisting of 93 women with a normal pelvis and 201 women with endometriosis. We measured plasma concentrations of interleukin (IL)-6, IL-8, tumour necrosis factor-alpha, high-sensitivity C-reactive protein (hsCRP), and cancer antigens CA-125 and CA-19-9. Analyses were done using the Kruskal-Wallis test, Mann-Whitney test, receiver operator characteristic, stepwise logistic regression and least squares support vector machines (LSSVM). RESULTS: Plasma levels of IL-6, IL-8 and CA-125 were increased in all women with endometriosis and in those with minimal-mild endometriosis, compared with controls. In women with moderate-severe endometriosis, plasma levels of IL-6, IL-8 and CA-125, but also of hsCRP, were significantly higher than in controls. Using stepwise logistic regression, moderate-severe endometriosis was diagnosed with a sensitivity of 100% (specificity 84%) and minimal-mild endometriosis was detected with a sensitivity of 87% (specificity 71%) during the secretory phase. Using LSSVM analysis, minimal-mild endometriosis was diagnosed with a sensitivity of 94% (specificity 61%) during the secretory phase and with a sensitivity of 92% (specificity 63%) during the menstrual phase. CONCLUSIONS: Advanced statistical analysis of a panel of six selected plasma biomarkers on samples obtained during the secretory phase or during menstruation allows the diagnosis of both minimal-mild and moderate-severe endometriosis with high sensitivity and clinically acceptable specificity.

Prevalent breast cancer patients with a homozygous mutant status for CYP2D6*4: response and biomarkers in tamoxifen users.

Retrospective studies suggest that single nucleotide polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene predict reduced tamoxifen metabolism, better tolerance and worse treatment outcome. We hypothesized that women with this genotype lack tamoxifen-induced endometrial and biochemical changes in follicle-stimulating hormone (FSH) and sex hormone-binding globulin (SHBG). We identified 56 breast cancer patients attending the follow-up clinic with a homozygous mutant (HM) status for the CYP2D6*4 null variant. Here, we report a detailed assessment of tamoxifen activity in 19 CYP2D6 HM women, while they were using tamoxifen either for metastatic (n = 5) or for early disease (n = 14). We assessed response to tamoxifen in metastatic disease. Endometrial appearances and serum levels of FSH and SHBG were assessed from retrospective and prospective testing. Our findings do suggest that the presence of two CYP2D6*4 alleles does not exclude a durable response of tamoxifen in metastatic breast cancer. The transvaginal ultrasonographic appearance of the endometrium in CYP2D6*4/*4 patients on tamoxifen is similar as seen in the normal population of tamoxifen users. The endometrium is increased in thickness with subepithelial cysts and endometrial polyps. Serum levels of FSH and SHBG in CYP2D6*4 HM tamoxifen users were in the range of what would be expected during tamoxifen treatment in the general population. Our findings do show CYP2D6*4/*4 carriers to have activity of tamoxifen on breast cancer, endometrium and serum levels of FSH and SHBG. They support clinical trials prospectively testing the effect of CYP2D6 genetic variability in response to tamoxifen before denying this drug to breast cancer patients only based on their CYP2D6*4 status.

The queen is dead--long live the workers: intraspecific parasitism by workers in the stingless bee Melipona scutellaris.

Insect societies are well known for their high degree of cooperation, but their colonies can potentially be exploited by reproductive workers who lay unfertilized, male eggs, rather than work for the good of the colony. Recently, it has also been discovered that workers in bumblebees and Asian honeybees can succeed in entering and parasitizing unrelated colonies to produce their own male offspring. The aim of this study was to investigate whether such intraspecific worker parasitism might also occur in stingless bees, another group of highly social bees. Based on a large-scale genetic study of the species Melipona scutellaris, and the genotyping of nearly 600 males from 45 colonies, we show that approximately 20% of all males are workers' sons, but that around 80% of these had genotypes that were incompatible with them being the sons of workers of the resident queen. By tracking colonies over multiple generations, we show that these males were not produced by drifted workers, but rather by workers that were the offspring of a previous, superseded queen. This means that uniquely, workers reproductively parasitize the next-generation workforce. Our results are surprising given that most colonies were sampled many months after the previous queen had died and that workers normally only have a life expectancy of approximately 30 days. It also implies that reproductive workers greatly outlive all other workers. We explain our results in the context of kin selection theory, and the fact that it pays workers more from exploiting the colony if costs are carried by less related individuals.

Effects of sublethal doses of crop protection agents on honey bee (Apis mellifera) global colony vitality and its potential link with aberrant foraging activity.

Honey bees (Apis mellifera) are the most economically valuable pollinators of fruit crops worldwide. Taking into account bees' contributions to other flowering agricultural crops, about one-third of our total diet comes directly or indirectly from bee-pollinated plants. However, in recent years there increasingly have been worrisome alarm sounds on serious bee mortalities and mysterious disappearance of bees from beehives. Among several environmental factors (e.g. climate and bee pathogens), stress factors arising from agricultural practices can potentially play a role in bee losses. Detailed knowledge on the effects of plant protection products is essential to improve usage with minimal risks. In order to identify potential medium- and long-term effects, we followed up various sublethal contaminated hives during the prolongation of the fruit-growing season. More specifically, a large-scale experiment was conducted in which at four distinct locations (in the Limburg region of Belgium) four different bee colonies (representing three different contaminations -imidacloprid, fenoxycarb, indoxacarb- and a non-contaminated control hive) were thoroughly monitored every 2-7 days. Our observations point towards decays of overall colony vitality for several hives a couple of weeks after treatment, as indicated by a set of carefully assessed parameters including the total amount of active and dead bees, total surface of capped brood and overall colony weight. These outcomes could be linked to subtle differences in foraging activity between distinct hives. The implications of these results are discussed in terms of potential short-term and long-term consequences of disturbed foraging ability triggered by exaggerated exposure to sublethal doses of crop protection chemicals, and its potential impact on colony health.

Egg marking in the facultatively queenless ant Gnamptogenys striatula: the source and mechanism.

Conflicts over reproductive division of labour are common in social insects. These conflicts are often resolved via antagonistic actions that are mediated by chemical cues. Dominant egg layers and their eggs can be recognized by a specific yet similar cuticular hydrocarbon profile. In the facultatively queenless ant Gnamptogenys striatula, a worker's cuticular hydrocarbon profile signals its fertility and this determines its position in the reproductive division of labour. How eggs acquire the same hydrocarbon profile is as yet unclear. Here, we search for glandular sources of egg hydrocarbons and identify the putative mechanism of egg marking. We found that eggs carry the same hydrocarbons as the cuticle of fertile workers, and that these hydrocarbons also occur in the ovaries and the haemolymph. None of the studied glands (Dufour, venom, labial and mandibular gland) contained these hydrocarbons. Our results indicate that hydrocarbons are deposited on eggs while still in the ovaries. The low hydrocarbon concentration in the ovaries, however, suggests they are produced elsewhere and transported through the haemolymph. We also found that fertile workers regularly deposit new hydrocarbons on eggs by rubbing laid eggs with a hairy structure on the abdominal tip from which a non-polar substance is secreted.

Amfor expression in the honeybee brain: a trigger mechanism for nurse-forager transition.

The honeybee's colony fitness relies on an optimized age-dependent division of labor. Transition from nursing activities to foraging activities is associated with an increase in the expression of the Amfor gene. Ben-Shahar et al. [Ben-Shahar, Y., Robichon, A., Sokolowski, M.B., Robinson, G.E., 2002. Influence of gene action across different time scales on behavior. Science 296, 741-744] showed that the Amfor transcripts and their gene products are involved in regulating the transition from one task to the next. In this study, we investigated the trajectory of the expression of this gene in the brain over time. The expression pattern could contribute to our understanding of the involvement of Amfor in the transition process. Is there a gradual increase in transcript or a peak in expression triggering a downstream path of multiple differential gene expression? Hereto, bees were sampled from colonies containing marked 1-day-old bees every 2 or 3 days around the expected time of transition from nurse to forager, from day 13 to 25. To quantify Amfor transcript in the brain, we developed a real-time RT-PCR assay, based on Taqman technology, using fluorescent probes. Results revealed a trigger mechanism rather than a continued elevation of Amfor expression. The appearance of an Amfor expression peak suggests that under normal physiological conditions foraging behavior is, at least in part, due to a trigger-effect of Amfor.

Free Testosterone Reflects Metabolic as well as Ovarian Disturbances in Subfertile Oligomenorrheic Women.

BACKGROUND: Diagnosing polycystic ovary syndrome (PCOS) is based on ovulatory dysfunction, ovarian ultrasound data, and androgen excess. Total testosterone is frequently used to identify androgen excess, but testosterone is mainly bound to sex hormone-binding globulin (SHBG) and albumin. Only 1-2% of nonprotein-bound testosterone (so-called free testosterone) is biologically active and responsible for androgen action. Moreover, automated immunoassays which are frequently used for female testosterone measurements are inaccurate. OBJECTIVE: To assess the clinical usefulness of liquid chromatography-tandem mass spectrometry measured testosterone and calculated free testosterone in subfertile women attending a fertility clinic with oligomenorrhea and suspected PCOS. METHODS: Hormonal and metabolic parameters were evaluated, and ovarian ultrasound was performed. Total testosterone was measured by liquid chromatography-tandem mass spectrometry. Free testosterone was calculated from total testosterone and SHBG. RESULTS: Sixty-six women were included in the study. Total testosterone was associated with ovarian volume and antral follicle count but not with metabolic parameters. However, SHBG and calculated free testosterone were associated with both ovarian ultrasound and metabolic parameters, such as BMI and insulin resistance. CONCLUSIONS: Assessing SHBG and free testosterone is important in evaluating androgen excess in subfertile women with ovulatory dysfunction and suspected PCOS, as it reflects both ovarian and metabolic disturbances.

Understanding and design of existing and future chromatographic support formats.

The present contribution reviews the use of alternative support formats as a means to surpass the chromatographic performance of the packed bed of spheres. First, a number of idealized structures are considered to obtain a general insight in how the performance of a chromatographic support depends on its shape and size, using the isocratic peak-capacity generation speed as the main performance indicator. Using this criterion, it is found that the packing density or, equivalently, the external porosity, is the most important of all geometrical shape factors. Depending on whether the sample consists of weakly or strongly retained components, the optimal external porosity can be expected to vary between 60% and a value near 100%. The optimal exploitation of a high external porosity, however, also requires overall shrinkage of the domain size, towards and into the sub-micron range. With the current fabrication technologies, this requirement seems difficult to achieve. In the presence of a lower limit on the characteristic support size, each range of desired plate numbers or peak capacities has its own optimal external porosity, ranging from a very low value (high packing density) for high speed, small peak capacity applications, to very high external porosities (low packing density) for applications requiring a very large peak capacity. Subsequently, the obtained theoretical insights are used to review and discuss the past and current research on alternative support formats. Finally, a number of emerging micro- and nano-fabrication technologies are introduced and their potential for the future production of supports with improved shape and homogeneity is discussed.

Influence of the packing heterogeneity on the performance of liquid chromatography supports.

We report on a series of plate height and flow resistance data obtained via computational fluid dynamics simulations in a simplified two-dimensional (2D) mimic of real packed bed and monolithic columns. By varying the external porosity (0.4 < epsilon < 0.8) and the degree of packing randomness, a good qualitative insight in the relationship between the packing porosity and heterogeneity and the general chromatographic performance parameters is obtained, unbiased by any differences in phase retention factor k', mobile phase diffusivity or viscosity or intra-skeleton porosity. The results provide a quantitative support for the use of domain size reduced plate heights as a means to compare the performance of chromatographic beds with a different porosity, as it was found that packings with a similar degree of packing heterogeneity yield very similar domain size reduced h(min)-values, nearly completely independent of the porosity. The study also clearly shows that the presence of preferential flow paths (inevitably accompanied by the presence of more clustered regions) leads to a decrease of the flow resistance, but also leads to a strong increase of the band broadening if supports with the same porosity epsilon and the same radial width are compared. For the presently considered 2D system, the flow resistance reduction is too small to overcome the corresponding strong increase in band broadening, such that the presence of preferential flow paths always leads to an overall increase of the separation impedance.

Calcium and bone homeostasis in heterozygous carriers of CYP24A1 mutations: A cross-sectional study.

BACKGROUND: Bi-allelic CYP24A1 mutations can cause idiopathic infantile hypercalcemia (IIH), adult-onset nephrocalcinosis, and possibly bone metabolism disturbances. It is currently unclear if heterozygous carriers experience clinical problems or biochemical abnormalities. Our objective is to gain insight in the biochemical profile and health problems in CYP24A1 heterozygotes. STUDY DESIGN: Cross-sectional evaluation of participants. Data of previously reported carriers are reviewed. SETTING AND PARTICIPANTS: Outpatient clinic of a tertiary care hospital. Participants were eight family members of an infant with a well-characterized homozygous CYP24A1 mutation c.1186C>T p.(Arg396Trp). OUTCOMES: Serum vitamin D metabolites. Symptoms or biochemical signs of hypercalcemia, hypercalciuria or nephrocalcinosis. Bone health in heterozygous as compared to wild type (WT) subjects. MEASUREMENTS: Genotyping by Sanger sequencing; vitamin D metabolites by liquid chromatography tandem mass spectrometry; renal, calcium and bone markers by biochemical analyses; presence of nephrocalcinosis by renal ultrasound; bone health by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. RESULTS: Six participants were heterozygous carriers of the mutation. None of the heterozygous subjects had experienced IIH. One had a documented history of nephrolithiasis, two others had complaints compatible with this diagnosis. No major differences between WT and heterozygous subjects were found regarding bone health, serum or urinary calcium or 25OHD/24,25(OH)2D ratio. Literature reports on three out of 33 heterozygous cases suffering from IIH. In all three, the 25OHD/24,25(OH)2D ratio was highly elevated. Nephrocalcinosis was frequently reported in family members of IIH cases. LIMITATIONS: Small sample size, lack of a large control group. CONCLUSIONS: Our and literature data suggest that most heterozygous CYP24A1 mutation carriers have a normal 25OHD/24,25(OH)2D ratio, are usually asymptomatic and have a normal skeletal status but may possibly be at increased risk of nephrocalcinosis. A review of the available literature suggests that an elevated 25OHD/24,25(OH)2D ratio may be associated with symptoms of IHH, irrespective of carrier status.

Particle counting immunoassay (PACIA). I. A general method for the determination of antibodies, antigens, and haptens.

By using a device designed for counting blood cells, it is possible to measure the agglutination of polystyrene beads (0.8 mu) with accuracy and great sensitivity, the agglutination resulting in a reduction in the number of particles. The latter coated with antigen can be used for determining IgM or IgG antibodies e.g. human rheumatoid factor or rabbit anti-bovine serum albumin antibodies. Macromolecules with multiple antigenic determinants agglutinate particles carrying specific antibodies. This system has been applied for determining HPL and alpha1-fetoprotein with a threshold of sensitivity of about 10 microgram/1. However the agglutination was decreased by serum factors which led to a 10-fold loss of sensitivity. The interference of rheumatoid factor which agglutinated the particles coated with rabbit or goat immunoglobulins could be avoided by reduction of the serum to be analyzed with 5mM dithiothreitol for 5 min. Haptens, i.e. DNP-lysine and T4, were determined by their inhibitory activities toward their specific antibodies, the agglutinator being a hapten-macromolecule conjugate or the antibodies themselves.

Deleterious Wolbachia in the ant Formica truncorum.

Wolbachia is a maternally inherited bacterium that may manipulate the reproduction of its arthropod hosts. In insects, it is known to lead to inviable matings, cause asexual reproduction or kill male offspring, all to its own benefit, but to the detriment of its host. In social Hymenoptera, Wolbachia occurs widely, but little is known about its fitness effects. We report on a Wolbachia infection in the wood ant Formica truncorum, and evaluate whether it influences reproductive patterns. All 33 colonies of the study population were infected, suggesting that Wolbachia infection is at, or close to, fixation. Interestingly, in colonies with fewer infected workers, significantly more sexuals are produced, indicating that Wolbachia has deleterious effects in this species. In addition, adult workers are shown to have significantly lower infection rates (45%) than worker pupae (87%) or virgin queens (94%), suggesting that workers lose their infection over life. Clearance of Wolbachia infection has, to our knowledge, never been shown in any other natural system, but we argue that it may, in this case, represent an adaptive strategy to reduce colony load. The cause of fixation requires further study, but our data strongly suggest that Wolbachia has no influence on the sex ratio in this species.

Widespread occurrence of the micro-organism Wolbachia in ants.

For more than 20 years, sex allocation in hymenopteran societies has been a major topic in insect sociobiology. A recurring idea was that relatedness asymmetrics arising from their haplodiploid sex determination system would lead to various parent-offspring conflicts over optimal reproduction. A possible weakness of existing theory is that only interests of nuclear genes are properly accounted for. Yet, a diversity of maternally transmitted elements manipulate the reproduction of their host in many solitary arthropod groups. The bacterium Wolbachia is a striking example of such a selfish cytoplasmic element, with effects ranging from reproductive incompatibility between host strains, induction of parthenogenesis and feminization of males. This paper reports on a first PCR-based Wolbachia screening in ants. Out of 50 Indo-Australian species, 50% screened positive for an A-group strain. One of these species also harboured a B-group strain in a double infection. Various factors that might explain the unusually high incidence of Wolbachia in ants are discussed. In general, Wolbachia may represent a widespread and previously unrecognized party active in the conflicts of interest within social insect colonies.