Consistency of dominance rank order: a comparison of David's Scores with I&SI and Bayesian methods in macaques.

In nonhuman primate social groups, dominance ranks are usually assigned to individuals based on outcomes of dyadic agonistic encounters. Multiple approaches have been used, but currently there is no consensus. One approach, David's Scores (DS), offers dual advantages of yielding cardinal scores that may in turn be used to compute hierarchical steepness. Here we correlate rank orders yielded by DS with those yielded by both the traditionally used I&SI approach and the recently proposed parametric Bayesian approach. We use six datasets for female macaques (three despotic and three tolerant groups), and 90 artificially generated datasets modeling macaque groups. We also use the artificial datasets to determine the impact of three characteristics (group size, interaction frequency, and directional asymmetry of aggression) on the magnitude of correlation coefficients, and assess the relative utility of two indices used to compute DS: Dij versus Pij. DS-based rank orders were strongly positively correlated with those yielded by the other two approaches for five out of the six macaque datasets, and for the majority of artificial datasets. Magnitudes of correlation coefficients were unrelated to group size or interaction frequency, but increased with directional asymmetry, suggesting methodological inconsistencies were more likely when dyads had more frequent reversals in directions of aggression. Finally, rank orders calculated using the Dij and Pij indices were similarly consistent with orders from other methods. We conclude that DS offers consistent estimates of rank orders, except perhaps in groups with very low levels of aggression asymmetry. In such "tolerant" groups, we suggest that the relatively greater methodological variability in rank orders may reflect behavioral characteristics of tolerant groups rather than computational inconsistencies between methods. We hypothesize that this quality may be quantified using posterior probability scores of Bayesian rank orders and may also index macaque social styles.

Bartonella species in bat flies (Diptera: Nycteribiidae) from western Africa.

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.

[Detection of porcine reproductive and respiratory syndrome virus (PRRS) genome fragments and anti-- bodies in chewing ropes allow -monitoring of PRRS in pig farms]. / Der Nachweis von Genomfragmenten des Porcinen Reproduktiven und ­Respiratorischen Syndrom Virus (PRRSV) und -spezifischen Antikörpern in Kaustricken ermöglicht die Überwachung PRRSV-unverdächtiger schweine­haltender Betriebe.

INTRODUCTION: Saliva samples from chewing ropes are a reliable diagnostic of porcine reproductive and respiratory syndrome virus (PRRSV) infections. The aim of this study was to test whether saliva samples taken with saliva swabs (cotton swabs and GenoTube Livestock) or with chewing ropes are suitable for monitoring PRRSV in unsuspicious farms, this means to detect a prevalence of 20% infected animals with a 95% probability. Saliva samples were collected from 12-16 pens in five pig farms by using a chewing rope for collective samples and by individual saliva swaps from five randomly selected animals per pen. A total of 291 animals from 58 pens in four study farms and 60 animals from 12 pens in one control farm were collected. The samples were taken from all age categories. According to the current monitoring system the analysis of five individual serum samples from the same pens served as the reference method for the relative sensitivity of the saliva samples. Serum and chewing rope samples were tested by ELISA for antibodies. Two different systems were used for the serum samples. Chewing ropes, saliva swabs (GenoTube Livestock) and serum samples were examined for virus genomes using a nested reverse-transcriptase PCR and a commercial real-time reverse-transcriptase PCR kit. Cohen's Kappa was used as a measure of agreement. PRRSV antibodies were detected in the chewing ropes of 44 pens and in the serum samples of only 34 pens. Viral RNA was found in 13 (chewing ropes), respectively 16 pens (serum samples). Saliva swabs (GenoTube Livestock) showed a lower relative sensitivity of 20.00% compared to serum samples. The agreement of the two serum analysis was very good for the ELISAs (κ = 0,911), and moderate for the PCR (κ = 0,706). The comparison of the chewing rope method with the analysis of the serum samples advocates this method as a suitable supplementary monitoring tool in PRRSV unsuspicious pig farms. Easy handling and lower examination costs of the chewing rope method allow higher testing frequency and would therefore improve the monitoring system. However, they are not an alternative to serum samples. Sampling with saliva swabs is unsuitable.

INTRODUCTION: Les échantillons de salive prélevés avec des cordes à mâcher ont fait leurs preuves dans la pratique pour diagnostiquer les infections à PRRSV. Le but de cette étude était de tester si des échantillons de salive prélevés avec des écouvillons salivaires (coton-tiges et GenoTube Livestock) ou avec des cordes à mâcher sont également adaptés au suivi des élevages non suspectés de PRRSV, c'est-à-dire de découvrir des animaux infectés avec une probabilité de 95% et une prévalence de 20%. Dans cinq exploitations, des échantillons de salive collectifs ont été prélevés dans 12 à 16 boxes à l'aide de cordes à mâcher et des échantillons de salive individuels provenant d'un échantillon aléatoire de cinq animaux par boxe ont été examinés. Un total de 291 animaux de 58 lots dans quatre exploitations d'étude et 60 animaux de 12 lots dans une ferme témoin ont été échantillonnés. Les échantillons ont été prélevés dans toutes les catégories d'âge. L'examen de cinq échantillons de sérum individuels provenant des mêmes lots sur la base d'un système de surveillance existant a servi de méthode de référence pour la sensibilité relative des échantillons de salive. Les échantillons de mastication et de sérum ont été testés pour les anticorps par ELISA en utilisant deux systèmes différents pour les échantillons de sérum. Les échantillons provenant des cordes à mâcher, les écouvillonnages de salive GenoTube Livestock et les échantillons de sérum ont été examinés à la recherche de génomes viraux à l'aide d'une PCR à transcriptase inverse emboîtée et d'un kit commercial de PCR à transcriptase inverse en temps réel. Le Kappa de Cohen a été utilisé comme mesure de concordance. À l'aide des cordes à mâcher, des anticorps PRRSV ont été détectés dans 44 enclos et à l'aide de sérum sanguin uniquement dans 34 enclos. L'ARN viral a été trouvé dans 13 (cordes à mâcher) et 16 (sérum) lots. Les écouvillons de salive GenoTube Livestock ont montré une sensibilité relative inférieure de 20,00% par rapport aux échantillons de sérum. La concordance des résultats de l'examen du sérum à l'aide de deux systèmes était très bonne pour les ELISA (κ = 0,911), pour les systèmes PCR modérée (κ = 0,706). La comparaison des échantillons issus de cordes à mâcher avec des échantillons de sérum montre qu'ils sont adaptés à une surveillance supplémentaire des élevages non suspectés d'être atteints du SDRPV. En raison de leur manipulation plus simple et de leurs coûts d'examen réduits, ils peuvent être utilisés pour augmenter la fréquence des examens et ainsi améliorer le système de surveillance, mais ils ne constituent pas une alternative aux échantillons de sérum.

In vivo evaluation of the uptake of [(123)I]FIAU, [(123)I]IVFRU and [(123)I]IVFAU by normal mouse brain: potential for noninvasive assessment of HSV-1 thymidine kinase gene expression in gliomas.

Radioiodinated 5-iodo-1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)uracil (F *IAU) is most commonly used for noninvasive assessment of herpes simplex virus type 1 thymidine kinase (HSV-1-tk) gene expression. However, it does not permeate the intact blood-brain barrier (BBB) because of its moderate lipophilicity. In this work, three iodo-nucleosides, FIAU, IVFRU, and IVFAU, were radiolabeled with iodine-123 and tested for permeation of the BBB in mice and for potential measurement of HSV-1-tk gene expression in gliomas. The results demonstrate that brain uptake and retention of these nucleosides is not directly related to their lipophilicity. The low brain uptake of IVFAU, in conjunction with its higher and constant brain/blood ratio, may reflect greater stability against hydrolysis of the N-glycosidic bond. In vivo PET evaluations of [(124)I]IVFRU and [(124)I]IVFAU in tumor-bearing mice are warranted.

Examples of in vivo isotype class switching in IgM+ chronic lymphocytic leukemia B cells.

Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5+ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur. To test the hypothesis that in vivo isotype class switching occurs in IgM+ B-type CLL cells, we analyzed the PBMC of 19 CLL patients for the presence of transcripts encoding the rearranged CLL V(H)DJ(H) associated with either gamma or alpha H chains. The molecular data indicate that approximately 50% of B-CLL patients have amplifications of IgM+ B cells that undergo an isotype class switch. Switching to IgA appears to occur more often than to IgG; also, switching can involve different IgG subclasses in individual patients. In many instances, these CLL-related gamma and alpha transcripts are much more plentiful than those of normal B cells that produce the same isotype. These switched transcripts do not reveal evidence for the accumulation of significant numbers of new V(H) gene mutations. The cellular data indicate that B cells with lesser amounts of surface membrane IgD and higher IgM/IgD ratios are more likely to undergo this switching process. Furthermore, B cells expressing IgG and IgA of the same idiotype or V(H) family and the same CDR3 length as those of the CLL IgM+ clone can be identified in the blood of patients studied using multiparameter immunofluorescence analyses. Collectively, these data suggest that not all members of a B-CLL clone are frozen at the surface membrane Ig-expressing stage of B cell maturation, and that some members can switch to the production of non-IgM isotypes. The occurrence of switching without the accumulation of V gene mutations indicates that the processes of differentiation and diversification are not linked.

Spidroins from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae).

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)n, (GGX)n, (GPGGX)n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.

Studies with Purified Chaperones Advance the Understanding of the Mechanism of Glucocorticoid Receptor-hsp90 Heterocomplex Assembly.

The study of the 9S, untransformed state of steroid receptors has led to the discovery of a multiprotein chaperone system that assembles heterocomplexes between hsp90 and a variety of proteins involved in signal transduction. Using the formation of glucocorticoid receptor (GR)-hsp90 heterocomplexes as a model, we have reconstituted a fully functional heterocomplex assembly system from purified components. The basic assembly system requires four proteins-hsp90, hsp70, p60/Hop and hsp40-to assemble GR-hsp90 heterocomplexes, which are then stabilized by the hsp90-interacting protein p23. The four proteins can self-assemble into an hsp90-p60/Hop-hsp70-hsp40 complex that we call a foldosome. Foldosomes isolated from reticulocyte lysate or formed from purified proteins open up a steroid-binding pocket to create a high-affinity steroid-binding state of the GR. We describe here the systematic reconstitution of the hsp90-based chaperone machinery and develop a model of the receptor-hsp90 heterocomplex assembly mechanism.

What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1ß, IL-8, LIF and TGFß2.

Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo.

A method has been developed that allows the isolation of genomic clones from a cosmid library by homologous recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into lambda phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologous plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 gene was restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively.

Cytoplasmic localization of the trypanothione peroxidase system in Crithidia fasciculata.

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.

Inefficiency of cynarin as therapeutic regimen in familial type II hyperlipoproteinaemia.

Seventeen ambulant outpatients with familial Type IIa or Type IIb hyperlipoproteinaemia were treated with Cynarin, the 1,5-dicaffeyl ester of quinic acid, the constituent of the artichoke (Cynara scolymus). The dose tested was 250 mg and 750 mg daily. The mean serum cholesterol and triglyceride concentrations were not significantly changed within 3 months. Cynarin, administered per os, has no hypolipidaemic effect in familial Type II hyperlipoproteinaemia.

Ability of various members of the hsp70 family of chaperones to promote assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90.

To be in a conformation that binds steroid, the hormone-binding domain of the glucocorticoid receptor (GR) must be bound to the 90 kDa heat shock protein (hsp90). Rabbit reticulocyte lysate contains a protein chaperone system that assembles the receptor into a heterocomplex with hsp90 and converts it from a non-steroid-binding to a steroid-binding form. Assembly of the GR-hsp90 heterocomplex requires hsp70, and in this work we examine the activities of four members of the hsp70 protein family in GR-hsp90 heterocomplex assembly. Rabbit reticulocyte lysate was depleted of hsp70 by passing it through a column of ATP agarose, resulting in the inactivation of its GR-hsp90 heterocomplex assembly activity. Addition of purified animal (mouse) or plant (wheat germ) hsp70 to the hsp70-depleted lysate permits assembly of a GR-hsp90 heterocomplex with a high affinity steroid binding site. However, purified hsp70 homologues from bacteria (DnaK) or the endoplasmic reticulum (BiP) do not promote heterocomplex formation, despite the fact that both DnaK and BiP bind to the GR in the assay system. When added to whole (i.e. hsp70-containing) reticulocyte lysate, DnaK and BiP inhibit GR-hsp90 heterocomplex assembly. Wheat germ lysate forms a heterocomplex between mouse GR and plant hsp90, but the addition of purified rabbit hsp70 to the wheat germ lysate does not increase the amount of receptor-wheat hsp90 complex produced, despite the fact that the rabbit hsp70 binds to the GR when it is added to the wheat chaperone system. The conclusion is that binding of hsp70 to receptors does not necessarily reflect a physiologically meaningful interaction. When native receptor heterocomplexes isolated from cytosols contain hsp70, it is likely that the hsp70-bound receptors represent a minority of receptors that have not yet proceeded fully through the receptor heterocomplex assembly process, which includes the dissociation of hsp70 after the binding of hsp90.

Highly complex genetic rearrangement involving at least seven breakpoints in a case of chronic myeloid leukemia.

Cytogenetic analysis for an atypical case of chronic myeloid leukemia (CML) showed a complex karyotype with four chromosome breakpoints (5q12, 12q21, 12q24, and 22q11) and translocation products that included a typical Philadelphia chromosome but apparently normal chromosomes 9. Molecular genetic analyses using four breakpoint cluster region (bcr) probes indicated that three breaks were probably present on chromosome 22. Two apparently independent breaks appeared to exist within the bcr, one of which was probably associated with a deletion of some bcr sequences. By combining the molecular and cytogenetic data, we could infer a total of seven breaks. This case illustrates the extensive and complex types of genetic alteration that may be associated with a c-abl and bcr fusion.

Spot synthesis of overlapping peptides on paper membrane supports enables the identification of linear monoclonal antibody binding determinants on morbillivirus phosphoproteins.

In order to map antigenic domains on the P-protein of morbillivirus, a series of overlapping peptides, representing the P-protein sequences of phocid distemper virus strain 2558/Han88 and canine distemper virus strain Onderstepoort, were synthesized on a paper support by the spot-technique. The reactivity of six monoclonal antibodies with the peptides was tested in an enzyme immunoassay and compared to their reactivity in Western blots and in an ELISA using detergent extracts from virus-infected cells. Three linear determinants could be localized on the P-protein. Two antibody-binding sites were delineated within the C-terminal (between amino acids 307-322 and 382-400, respectively), and a third one was located on the N-terminal part (amino acids 13-31) of the protein. Fine mapping of this binding site revealed that this was a part of an antigenic domain. In Western blots, the monoclonal antibodies reacting with this domain also reacted with a second protein which was possibly the V-protein.

Secretion of glycosylated human interleukin-2 by recombinant mammalian cell lines.

The production of glycosylated forms of the human T cell growth factor (interleukin-2, IL-2) has been studied after transfection of a mouse L cell line and a chinese hamster ovary cell line with a plasmid containing the human chromosomal interleukin-2 gene. Both cell lines produced IL-2 constitutively. Based on their behavior in reversed-phase l.c. and their sodium dodecyl sulfate-gel-electrophoresis pattern, human IL-2 protein secreted by L cells showed a similar distribution of glycosylated (Mr 16 500) and nonglycosylated (Mr 14 500) forms as the natural protein secreted by human peripheral lymphocytes, whereas the hamster cell line secreted preponderantly the glycosylated forms. Exoglycosidase digestion of the 16 500 Mr IL-2 protein shifted the gel electrophoretic mobility towards the low-molecular weight form as is true for the natural glycosylated IL-2, which contains the usual tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-[alpha-NeuAc-(2----6)]-D-GalNAc (IL-2 N2) and the trisaccharide alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-D-GalNAc (IL-2 N1) as the major carbohydrate constituents. These results support the applicability of recombinant DNA technology as a tool for studying glycoprotein biosynthesis in mammalian cells.

Myxothiazol: a reversible blocker of the cell cycle.

Myxothiazol, a potent inhibitor of the cytochrome bc1 oxidoreductase, was shown by the use of flow cytometry to block reversibly the late G1/S phase of the cell cycle of human lymphoblastic T-cell line Jurkat (clone 886) at concentrations of 0.5 microgram/ml. These observations are compared to those of other drugs, such as antimycin, which effect the respiratory chain, and with O2-deficiency.

Paleoparasitological analysis of coprolites from K2, an Iron Age archaeological site in South Africa: the first finding of Dicrocoelium sp. eggs.

Until now, Dicrocoelium sp. eggs have only been recorded from European and 1 North American archaeological sites. We present evidence for the first record of Dicrocoelium sp. from an African archaeological site. A paleoparasitological study was conducted on 7 coprolite samples from K2, a Late Iron Age site on the farm Greefswald, in the Northern Province of South Africa. Standard parasitological analysis revealed the presence of Dicrocoelium sp. and Trichuris sp. eggs. Today, the parasite does not occur in this region. Trichurid eggs are a relatively common find in paleoparasitological analysis. The presence of Dicrocoelium sp. provides new clues about the antiquity of this parasite, as well as aspects of ancient environment, climate, and interactions among humans, animals, and parasites.

Crab louse infestation in pre-Columbian America.

Until now, Pthirus pubis infestation in ancient human populations had only been recorded in the Old World. We found crab lice on South American mummified bodies from the Atacama Desert region. Crab louse eggs were found attached to the pubic hairs of a 2,000-yr-old Chilean mummy. Well-preserved adults were found in sediment and clothing from a Peruvian mummy dated 1,000 yr ago. Paleoparasitological evidence expands the knowledge of the distribution of this ectoparasite in ancient populations. As with many other parasites, pubic lice recorded in Andean populations show the antiquity of this parasite in the New World. It is likely that P. pubis entered the continent with early human migration to the New World.

Assessment of the relative number of copies of the gene encoding human neutrophil antigen-2a(HNA-2a), CD177, and a homologous pseudogene by quantitative real-time PCR.

Human neutrophil antigen-2a (HNA-2a; NB1) is located on the 58-64 kD NB1 glycoprotein (GP) and is encoded by the gene CD177. Searches of human genome databases have revealed that a pseudogene highly homologous to exons 4-9 of CD177 is located adjacent to CD177 on chromosome 19. The purpose of this study was to document the presence of the pseudogene and determine whether the polymorphic expression of NB1 GP is due to CD177 gene deletions and duplications. Genomic DNA was isolated from leukocytes of 12 subjects. The number of copies of exon 2 of CD177, an exon that is unique to this gene, and the number of copies of exon 9, an exon that is found in both CD177 and the pseudogene, was assessed with quantitative real-time PCR. The ratio of the number of copies of sequences homologous to CD177 exon 9 to the number of copies of exon 2 was 1.5 or greater in 7 of the 12 subjects, suggesting that both CD177 and the homologous pseudogene were present. The ratio of exon 9 to exon 2 in the other 5 subjects ranged from 1 to 1.25, suggesting that the pseudogene was not present in these subjects. However, results of assays were variable and we could not exclude the possibility that all subjects carried the pseudogene. These studies confirmed the presence of the pseudogene homologous to CD177, but quantitative real-time PCR was not precise enough to detect CD177 duplications or deletions.