IntroductionThe Portuguese donor Registry of CEDACE was the fifth largest per capita bone marrow donor Registry of the WMDA as of 2019 and has yet to be thoroughly analyzed. We aimed to characterize its various aspects, including demographics and HLA allele and haplotype frequencies, to evaluate the genetic matching propensity score and ultimately further develop it.MethodsWe described and compared characteristics of the donor population with census data and used an Expectation-Maximization algorithm and analyses of molecular variance to assess haplotype frequencies and establish phylogenetic distances between regions and districts within the country.ResultsWe identified 396545 donors, corresponding to 3.85% of the Portuguese population; the median donor age was 39 years, with 60.4% of female donors. Most donors were Portuguese nationals, although 40 other nationalities were present, with a significant proportion of donors from Brazil and Portuguese-speaking African Countries; almost all donors self-reported as Western, with the second largest group reporting African ancestry. There was an asymmetric contribution of donors from different districts and regions, with most coming from coastal districts and few from the southern districts and autonomous regions; foreign and self-declared non-Western donors were mainly located in the Metropolitan Area of Lisbon and the South. Although most donors were typed in three loci (HLA-A, HLA-B and HLA-DRB1), only 44% were also typed in HLA-C, 1.28% in HLA-DQB1 and only 0.77% in all five loci and in high-resolution. There were varying allele and haplotype frequencies across districts and regions, with the most common three loci, low-resolution haplotypes, being HLA-A*01~B*08~DRB1*03, A*29~B*44~DRB1*07 and HLA-A*02~B*44~DRB1*04; some haplotypes were more prevalent in the South, others in the North and a few in the autonomous regions; African and foreign donors presented relevant differences in haplotype frequency distributions, including rare haplotypes of potential interest. We also report on four loci, low-resolution frequency distributions. Using AMOVA, we compared genetic distances between districts and regions, which recapitulated the country's geography.DiscussionOur analysis showed potential paths to optimization of the Registry, including increasing the male donor pool and focusing on underrepresented districts and particular populations of interest, such as donors from Portuguese-speaking African countries.

The taxonomic identity of Mentha species is difficult as it displays variations at morphochemical characters. The aim of this investigation was to study Mentha spp. diversity in 60 Tunisian accessions using morphological traits and molecular markers. Thirty-three botanical descriptors were analysed. The numerical taxonomic analysis identified 58 accessions and defined seven taxonomic groups including M. aquatica, M. longifolia, M. piperita, M. pulegium, M. rotundifolia, M. spicata and M. spicata var. crispa 'moroccan'. Thereafter, genetic diversity of these accessions was investigated using ISSR markers. Nine highly polymorphic ISSR primers were selected to amplify DNA of each accession. Based on UPGMA method, these accessions could be grouped into two major clusters. M. pulegium group occupied a relatively basal position in the dendrogram and formed a single cluster. The second large cluster contained the accessions of the section Mentha, clustered into five subgroups. Molecular separation of the Mentha suggested that hybridisation among species may result in convergence and/or divergence of different species. Our study has shown (1) a high morphological and molecular diversity in the Tunisian accessions and (2) that combining morphological and molecular data is crucial to a proper identification, allowing the correct use of these accessions in future breeding programmes.

OBJECTIVE:To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in systemic sclerosis (SSc) patients.METHODS:Spontaneous production of CXCL10, CCL4, CXCL8 and IL-6 was intracellularly evaluated in classical, non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood of SSc patients and healthy controls (HC) through flow cytometry. In addition, production of these cytokines was determined upon toll-like receptor (TLR) 4 plus Interferon-γ (IFN-γ) stimulation.RESULTS:The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both limited (lcSSc) and diffuse cutaneous (dcSSC) subsets of SSc patients and CCL4 was augmented in dcSSc patients. The proportion of CCL4-producing mDCs was also elevated in dcSSc patients and the percentage of mDCS producing CXCL10 only in lcSSc patients. Upon stimulation, the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups and mDCs expressing CXCL8 only in lcSSc. Moreover, these parameters in unsupervised clustering analysis identify a subset of patients which are characterized by lung fibrosis and reduced pulmonary function.CONCLUSIONS:These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis.

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8 + T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4 + T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.

Programmed cell death-1 protein (PD-1) is an immune checkpoint that has gained popularity in the treatment of several advanced cancers. Inhibiting this checkpoint is known to enhance immune response, but is also known to diminish immune tolerance and to increase autoimmune toxicity. We discuss a case of rapid onset fulminant Type 1 diabetes induced by treatment with anti-programmed cell death-1 monoclonal antibody, nivolumab, in a patient with late-stage non-small-cell lung adenocarcinoma. The patient had no history of previous diabetes but did reveal a high-risk genotype for Type 1 diabetes development (DR3-DQ2; DR4-DQ8). This finding supports that acute Type 1 diabetes can be an important adverse effect of immunotherapies targeting T-cell activation regulation. Because of the severity of this adverse effect, physicians should be aware of it, and studies directed to the detection of new biomarkers for early risk stratification (e.g., HLA) should be sought.

Neonates and small infants with syndromes characterized by the presence of craniofacial abnormalities may represent great challenges regarding the management of the airway. We describe the case of a 9-day-old neonate with Treacher Collins syndrome, in which a laryngeal mask was essential to improve the airway obstruction, ventilate the patient and serve as an airway conduit for a fiberoptic intubation. By presenting this case, we intend to show that in neonates with Treacher Collins syndrome, in whom difficulties ventilation and intubation are expected, a thoughtful airway management planning is mandatory.

1Grupo Português Génito-Urinário, Hospital São José, 1050-059 Lisboa, Portugal. 2UCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 3Centro de Estudos de Doenças Crónicas, CEDOC, NOVA Medical School/Faculdade de Ciências Médicas, Universidade Nova de Lisboa, 1169-056 Lisbon, Portugal. 4CDG & Allies, Professionals and Patient Associations International Network (CDG & Allies PPAIN), Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 5Centro de Sangue e Transplantação de Lisboa, IPST, IP., 1749-005 Lisboa, Portugal. 6EAU Guidelines Office, 24 1040 Brussels, Belgium.

Background Systemic sclerosis (SSc) is an inflammatory and fibrotic disease characterized by vascular dysfunction, excessive extracellular matrix deposition and immune dysregulation. Recent observations suggest that monocytes and dendritic cells (DCs) might be involved in SSc; including cell recruitment, trafficking, activation and an enhanced pro-fibrotic phenotype. Hence these cells might be important contributors to the disease pathogenesis. However, detailed analysis of circulating monocytes and DCs in SSc in relationship to disease activity has not been performed so far. Objectives To investigate the ex vivo pro-inflammatory properties of classical and non-classical monocytes as well as myeloid dendritic cells (mDCs) in SSc patients in relationship to disease activity. Methods This study enrolled 43 SSc patients, 30 classified as limited cutaneous SSc (lcSSc) and 13 as diffuse cutaneous (dcSSc). The healthy control group (HC) included 20 age- and gender- matched individuals. The Spontaneous production of CXCL10, CCL4, CXCL4 and IL-6 was intracellularly evaluated in classical and non-classical monocytes and Siglec-3-expressing mDCs from peripheral blood using flow cytometry. In addition, the production of these cytokines was determined upon toll like receptor 4 (TLR4) plus Interferon-γ (IFN-γ) in vitro stimulation. Results The frequency of non-classical monocytes spontaneously producing CXCL10 was increased in both lcSSc and dcSSC subsets of SSc patients (p<0.05) and CCL4 was augmented in the dcSSc patient subset (p<0.05). The proportion of CCL4 producing- mDCs were also elevated in dcSSc patients (p<0.01) and the percentage of mDCS producing CXCL10 only in lcSSc patients (p<0.05 compared to HC, but p<0.01 comparing to dcSSc). Upon in vitro stimulation the frequency of non-classical monocytes expressing CXCL8 was increased in both patient groups (p<0.01) and mDCs expressing CXCL8 only in lcSSc (p<0.01). SSc patients characterized by the presence or history of lung fibrosis, displayed a higher frequency of non-classical monocytes expressing CCL4 and CXCL10 in dcSSc patients as compared to those without this clinical manifestation (p<0.01 and p<0.05 respectively). Strikingly, the percentage of classical monocytes producing CXCL8 was augmented upon in vitro stimulation in lcSSc patients with lung fibrosis as compared to those without (p<0.01). No differences were found in the percentage of IL-6 producing cells. Conclusions These data point towards a role of activated non-classical monocytes and mDCs producing enhanced levels of proinflammatory cytokines in SSc, potentially contributing to lung fibrosis. Acknowledgements TC is supported by a grant from the Portuguese national funding agency for science, research and technology: Fundação para a Ciência e a Tecnologia [SFRH/BD/93526/2013]. Disclosure of Interest None declared

The diversity of killer-cell immunoglobulin-like receptors (KIR) genes was evaluated in Portuguese and the observed genotypic profiles were found related to the ones reported in European populations. The KIR repertoire after hematopoietic stem cell transplantation is determined by these gene frequencies and the KIR group B motifs are the less common. We estimated donor-KIR/recipient-ligand interactions in transplants with related donors and unrelated donors found in a local registry or from abroad. A large fraction of transplants had all three ligands of inhibitory receptors, and therefore, in theory were not prone to natural killer cell (NK) mediated alloreactivity. Furthermore, the distribution of KIR alloreactive interactions was found independent of the donor-recipient genetic proximity, probably because of different gene segregation and comparable KIR frequencies in the donor pools.

In view of its heterogeneous presentation and unpredictable course, clinical management of systemic lupus erythematosus (SLE) is difficult. There is a need for biomarkers and diagnostic aids to monitor SLE disease activity and severity prior to, during and after treatment. We undertook this study to search for unique phenotypic patterns in each peripheral blood (PB) B cell subset, capable of distinguishing SLE patients with inactive disease versus SLE patients with active disease versus controls by using an automated population separator (APS) visualization strategy. PB was collected from 41 SLE patients and 28 age- and gender-matched controls. We analyzed the cell surface markers (in a tube CD20/CD27/CD19/CD45/CD38/CD81/BAFFR combination) expression on PB B cell subsets using principal component analysis, implemented in the APS software tool. Overall, our analysis indicates that active SLE can be distinguished from inactive SLE on the basis of a single tube analysis, focused on the decreased expression of CD38, CD81 and BAFFR in transitional B cells. The cluster analysis of immunophenotypic profiles of B cell subsets highlighted disease-specific abnormalities on transitional B cells that emerge as promising surrogate markers for disease activity. Further validation is needed with larger samples and prospective follow-up of patients.

Systemic inflammation has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD) systemic effects. However, most COPD patients do not suffer from persistent systemic inflammation even after exacerbations and exercise and scientific evidence has provided conflicting results. Our aim is to evaluate inflammatory gene expression at rest and at 1 and 24 h after strenuous exercise in COPD patients and study the patient variables associated with inflammatory expression. A cross-sectional study was conducted in COPD patients who were recruited on entry to a pulmonary rehabilitation (PR) program. Demographic, clinical and functional data were collected. Blood samples were collected and gene expression was analyzed by reverse transcriptase polymerase chain reaction for IFNg, IL1b, IL6, IL8, TNFa, TGFb1 and iNOS. The study included 21 patients (15 men, 71.4%), mean age 66.1 years old (SD = 8.27), mean FEV1 46.76% (SD 20.90%), 67% belonging to GOLD grade D, mean BODE index of 3.9, 90.5% with smoking history, mean BMI 25.81 (SD = 4.87), median of 1.29 exacerbations in the previous year. There was no statistical significant difference between inflammatory expression at rest and at 1 h and 24 h after the maximal exercise test for all tested genes. We found an association between BMI and inflammatory expression at all the points of time checked, a slight inverse association occurs with low BMI for mRNA IL1b, IL6, TNFa, TGFb1 and iNOS, and there was a more pronounced positive association for obese patients for all tested genes. This preliminary study did not show an enhanced inflammatory gene expression from rest to 1 h and 24 h after short-term exercise, but did show an increased inflammatory gene expression in both BMI extremes, both at rest and after exercise, suggesting not only malnourishment, but also obesity as potential links between COPD and systemic inflammation. Studies with larger samples and designed to definitely exclude OSA or OHS as confounding factors in obese patients are required.

The polymorphism of HLA genes can be used to reconstruct human peopling history. However, this huge diversity impairs successful matching in stem cell transplantation, a situation which has led to the recruitment of millions of donors worldwide. In parallel to the increase of recruitment, registries are progressively relying on information from population genetics to optimize their donor pools in terms of HLA variability. In this study, the HLA data of 65,000 Spanish bone marrow donors were analyzed together with 60,000 Portuguese individuals to provide a comprehensive HLA genetic map of the Iberian Peninsula. The frequencies of many alleles were shown to vary continuously across the Peninsula, either increasing or decreasing from the Mediterranean coast to the Atlantic domain or from the Strait of Gibraltar to the Pyrenees and Bay of Biscay. Similar patterns were observed for several haplotypes. In addition, within some regions neighboring provinces share a close genetic similarity. These results outline the genetic landscape of the Iberian Peninsula, and confirm that the analysis of the HLA polymorphism may reveal relevant signatures of past demographic events even when data from donor registries are used. This conclusion stimulates future developments of the Spanish registry, presented here for the first time.

Here, we evaluated the distribution and functional profile of circulating CD27+ and CD27− γδ T-cell subsets in systemic sclerosis (SSc) patients to assess their potential role in this disorder.

Systemic Lupus Erythematosus (SLE) is an auto-immune disease whose complex pathogenesis remains unraveled. Here we aim to explore the inflammatory ability of SLE patients’ sera upon peripheral blood (PB) monocyte subsets and myeloid dendritic cells (mDCs) obtained from healthy donors.

OBJECTIVES AND DESIGN:Here we evaluated whether allergic rhinitis to Dermatophagoides pteronyssinus induces alterations on circulating B cell subsets.MATERIALS AND METHODS:Circulating B cell subsets and isotype expression on antigen-experienced B cells from allergic patients under conventional pharmacological treatment (NO-SIT, n = 15) and under subcutaneous immunotherapy (SCIT, n = 33), and non-allergic subjects (NC, n = 25) were analyzed by flow cytometry.RESULTS:In allergic patients, we found a significant decrease in IgM(+) and IgG(+) memory B cells and an increase in IgA(+) memory B cells. Additionally, the numbers of circulating IgA(+) plasmablasts in allergic patients were also increased, while those cells expressing IgM were reduced.CONCLUSIONS:Allergic patients have a disturbed B cell subsets distribution which seems to underlie rhinitis pathogenesis and remain unchanged after SCIT.