Ana Elisabete Pires

Last lynxes in Portugal? Molecular approaches in a pre-extinction scenario

The Iberian lynx is the most threatened felidin the world and has suffered a declinethroughout its range. Effective monitoring ofthe species presence is essential. Fieldwork inpreviously identified areas of lynx occurrencein Portugal has resulted in the collection of104 possible lynx scats. Recently, there hasbeen little or no evidence of lynx presence andscats could be confused with others from moreabundant carnivores such as wildcat, fox anddog. In order to confirm or not exclude thepresence of the species, identification ofscats was performed through the amplificationof lynx-specific mitochondrial DNA sequences.Two samples collected in Malcata NaturalReserve in 1997 were identified as lynx. Thisis the most recent and reliable proof of lynxpresence in Portugal*. Given the territorialbehavior of lynx, stable resident populationswould have produced a higher proportion ofpositively identified scats. Local extinctionsmight have taken place, and this genetic datasupports a suspected national pre-extinctionscenario for the species. Genetic analysisusing a non-invasive approach has proved to bean informative part of the lynx monitoringprogram. Technical problems faced and overcomeare also presented.

Broadened T-cell Repertoire Diversity in ivIg-treated SLE Patients is Also Related to the Individual Status of Regulatory T-cells

PurposeIntravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus, the mechanism of which remains to be fully understood. Here we investigated whether ivIg affects two established sub-phenotypes of SLE, namely relative oligoclonality of circulating T-cells and reduced activity of CD4u2009+u2009Foxp3+ regulatory T-cells (Tregs) reflected by lower CD25 surface density.MethodsWe conducted a longitudinal study of 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2–6 consecutive monthly infusions. Among these 15 patients, 10 responded to ivIg therapy with clear clinical improvement. We characterized Tregs and determined TCR spectratypes of four Vβ families with reported oligoclonality. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vβ-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points.ResultsFor 11 out of 15 patients, average Vβ1/Vβ2/Vβ11/Vβ14 repertoires were less perturbed under than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients Treg CD25 surface density (cytometric MFI) was clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients average Treg CD25 MFI was found negatively correlated with both Vβ11 and Vβ14 perturbations measured under ivIg therapy.ConclusionsThis indicates a role of active Tregs in the therapeutic effect of ivIg.

Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for approximately 30% of the genetic variation. However, analysis of the genetic structure in non-cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.

Treatment With Polyclonal Immunoglobulin During T-cell Reconstitution Promotes Naive T-cell Proliferation

Natural antibodies are unique self molecules endowed with both suppressive and activating functions on various cells of the immune system and are recognized as a fundamental link between the adaptive and innate immune system. Here, we examine the role of natural antibodies, using polyclonal immunoglobulins (Ig), as a promoter of T-cell reconstitution in a context of lymphopenia. We have established a mouse model to mimic immunologic recovery in adult patients with severe hypothymic function subjected to autologous hematopoietic precursor cell transplantation. Thymectomized mice were transplanted and treated with low doses of Ig or its Fab or Fc fragments. The animals displayed, during early phases of Ig treatment, a significant increase of T-cell reconstitution displaying a naive CD4+phenotype. In addition, the Ig-treated animals exhibited an increase dilution of single-joint T-cell receptor excision circles (sjTRECs) in peripheral blood, suggesting an early increase in proliferation of T cells stimulated by the natural antibodies. These results unveil a novel and considerable effect of intravenous Ig treatment in situations of severe lymphopenia as a stimulator of proliferation of peripheral naive T cells, possibly protecting diverse immune repertoires.

Treatment with Low Doses of Polyclonal Immunoglobulin Improves B Cell Function During Immune Reconstitution in a Murine Model

ProposeAfter autologous stem cell transplantation (ASCT) the immunological B cell compartment recovers slowly. Delays on the recovery of B cell function after autologous stem cell transplantation are due to the low lymphocytes count and to their intrinsic dysfunction.MethodsWe studied the in vivo B cell reconstitution after ASCT examining the independent effect of polyclonal IgG (PolyIg), Fab or Fc fragments infusions in a murine animal model during a period of 12xa0weeks. These molecules were used in low doses, mimicking the recommended use of IVIg in the case of hypogammaglobulinemia in humans. Flow cytometry analysis and ELISA tests were conducted to monitor the reconstitution of B cells and serum immunoglobulin production. Panama blot and PCA factor 1 analysis were used to study the kinetics of immunoglobulin repertoires reconstitution. Mechanistic studies were also performed using in vitro cell culture.ResultsDuring follow-up after ASCT, peripheral B cells expand independently of treatment, correcting the immediate increase in sBAFF (soluble B cell activating factor) induced by previous intense myeloablation. Treatments with Fab and Fc fragments infusions promote significant IgM and IgG production comparing to control. Although the complete recovery of antibody repertoire is only achieved at the end of follow-up after ASCT, there is an earlier and significantly stronger recovery in the treated mice, which is evident at 9xa0weeks after ASCT. At 30xa0weeks after ASCT, normal values of antibody repertoire were detected in all individuals. Mechanistic studies show that Fab and Fc fragments promote IgG1 production by indirect pathways.ConclusionsThe results presented here demonstrate that polyclonal immunoglobulin indirectly improves the function of the reconstituted B cells and their IgG production by means of Fc-mediated effects on bystander cells. These results further stimulate the discussion about the advantages of IVIg therapy during immune reconstitution after human ASCT.

New insights into the genetic composition and phylogenetic relationship of wolves and dogs in the Iberian Peninsula

Abstract This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.

Active regulatory T-cells contribute to broadened T-cell repertoire diversity in ivIg-treated SLE patients

Intravenous IgG (ivIg) is a therapeutic alternative for lupus erythematosus. Relative oligoclonality of circulating T-cells in SLE has been reported. Also CD4+Foxp3+ regulatory T-cells (Tregs) have a characteristically reduced activity in SLE, reflected by CD25 surface density. Aiming to study the role of Tregs for ivIg therapy, we characterized Tregs and determined TCR spectratypes of four Vb families with reported oligoclonality, in 15 lupus patients (14 with SLE and one with discoid LE) treated with ivIg in cycles of 2-6 consecutive monthly infusions. Among these 15 patients, 11 responded with clinical improvement. Cell counts, cytometry and TCR spectratypes were obtained from peripheral blood at various time points before, during and after ivIg treatment. T-cell oligoclonality was assessed as Vb-familywise repertoire perturbation, calculated for each patient in respect to an individual reference profile averaged over all available time points. For 11/15 patients, average Vb1/Vb2/Vb11/Vb14 repertoires were less perturbed under ivIg treatment than outside ivIg therapy. The four exceptions with relatively increased average perturbation during ivIg therapy included three patients who failed to respond clinically to an ivIg therapy cycle. Patients Treg CD25 surface density (cytometric MFI) was, other than Treg/CD4+ frequency, clearly reduced when compared to healthy controls, but not obviously influenced by ivIg. However, patients average Treg CD25 MFI was found negatively correlated with both Vb11 and Vb14 perturbations measured under ivIg therapy, which indicates a role of active Tregs in the therapeutic effect of ivIg.

Reinforcement of Thermoplastic Corn Starch with Crosslinked Starch/Chitosan Microparticles

Microparticles of corn starch and chitosan crosslinked with glutaraldehyde, produced by the solvent exchange technique, are studied as reinforcement fillers for thermoplastic corn starch plasticized with glycerol. The presence of 10% w/w chitosan in the microparticles is shown to be essential to guaranteeing effective crosslinking, as demonstrated by water solubility assays. Crosslinked chitosan forms an interpenetrating polymer network with starch chains, producing microparticles with a very low solubility. The thermal stability of the microparticles is in agreement with their polysaccharide composition. An XRD analysis showed that they have crystalline fraction of 32% with Va-type structure, and have no tendency to undergo retrogradation. The tensile strength, Young’s modulus, and toughness of thermoplastic starch increased by the incorporation of the crosslinked starch/chitosan microparticles by melt-mixing. Toughness increased 360% in relation to unfilled thermoplastic starch.

Cremation under fire: a review of bioarchaeological approaches from 1995 to 2015

The study of bioarchaeological evidence associated with burials is essential for achieving a global perspective on cremation as a funerary practice, its chronological and geographical distribution, as well as its inner socio-cultural and technological diversity. However, for that purpose, similar and consistent analyses must be adopted by bioarchaeologists to enable intra- and inter-sites comparisons. The 1995–2015 literature encompassing 84 geographically representative articles concerning bioarchaeological studies of burned human skeletal remains is reviewed herein. The objective was to assess methodological variability. Information concerning colour, fragmentation, skeleton completeness, ‘skeletal region’ representation, non-human funerary assemblage, pre-burning condition of the remains, minimum number of individuals, biological profile, trauma and pathologies was considered. The results demonstrate that certain methods were used by almost all researchers. That was the case for colour description (91xa0%), skeleton completeness (91xa0%), minimum number of individuals (96xa0%), age-at-death (100xa0%) and sex of the individuals (95xa0%). Researchers are much more divided about the implementation of the remaining methods. Methodological choices also vary. The asymmetries in the selection of the analyses that are undertaken can lead to different interpretations and conclusions of the contexts under study. This may prevent consistent comparisons within the same site and between different sites. We emphasize the need for bioarchaeologists to discuss and standardize analytical procedures for studying cremated remains.