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The rapid development of whole genome sequencing technologies and the subsequent integration of pathogen and human genomic information into routine public health activities has the potential to benefit the population-level management of infectious diseases. The comprehensive description of the current landscape of infectious disease surveillance, the identification of priority pathogens, and the assessment of the perception and needs of a wide range of stakeholders provide a solid base to guide the implementation of pathogen genomics. Subsequent pathogen-specific study cases within a real-world public health setting enabled to demonstrate the added value and pointed important challenges for the integration of genomic information in epidemiological analyses. As such, the findings of this thesis can further guide the integration of genomic information for the surveillance and control of infectious diseases within the national public health institute in Belgium., (SP - Sciences de la santé publique) -- UCL, 2022

Dependence on multiple nutritional bacterial symbionts forming a metabolic unit has repeatedly evolved in many insect species that feed on nutritionally unbalanced diets such as plant sap. This is the case for aphids of the subfamilies Lachninae and Chaitophorinae, which have evolved di-symbiotic systems in which the ancient obligate nutritional symbiont Buchnera aphidicola is metabolically complemented by an additional nutritional symbiont acquired more recently. Deciphering how different symbionts integrate both metabolically and anatomically in such systems is crucial to understanding how complex nutritional symbiotic systems function and evolve. In this study, we sequenced and analyzed the genomes of the symbionts B. aphidicola and Serratia symbiotica associated with the Chaitophorinae aphids Sipha maydis and Periphyllus lyropictus. Our results show that, in these two species, B. aphidicola and S. symbiotica complement each other metabolically (and their hosts) for the biosynthesis of essential amino acids and vitamins, but with distinct metabolic reactions supported by each symbiont depending on the host species. Furthermore, the S. symbiotica symbiont associated with S. maydis appears to be strictly compartmentalized into the specialized host cells housing symbionts in aphids, the bacteriocytes, whereas the S. symbiotica symbiont associated with P. lyropictus exhibits a highly invasive phenotype, presumably because it is capable of expressing a larger set of virulence factors, including a complete flagellum for bacterial motility. Such contrasting levels of metabolic and anatomical integration for two S. symbiotica symbionts that were recently acquired as nutritional co-obligate partners reflect distinct coevolutionary processes specific to each association.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Many insect species are associated with bacterial partners that can significantly influence their evolutionary ecology. Compared to other insect groups, aphids harbor a bacterial microbiota that has the reputation of being poorly diversified, generally limited to the presence of the obligate nutritional symbiont Buchnera aphidicola and some facultative symbionts. In this study, we analyzed the bacterial diversity associated with the dogwood-grass aphid Anoecia corni, an aphid species that spends much of its life cycle in a subterranean environment. Little is known about the bacterial diversity associated with aphids displaying such a lifestyle, and one hypothesis is that close contact with the vast microbial community of the rhizosphere could promote the acquisition of a richer bacterial diversity compared to other aphid species. Using 16S rRNA amplicon Illumina sequencing on specimens collected on wheat roots in Morocco, we identified 10 bacterial operational taxonomic units (OTUs) corresponding to five bacterial genera. In addition to the obligate symbiont Buchnera, we identified the facultative symbionts Serratia symbiotica and Wolbachia in certain aphid colonies. The detection of Wolbachia is unexpected as it is considered rare in aphids. Moreover, its biological significance remains unknown in these insects. Besides, we also detected Arsenophonus and Dactylopiibacterium carminicum. These results suggest that, despite its subterranean lifestyle, A. corni shelter a bacterial diversity mainly limited to bacterial endosymbionts.

Coronavirus disease 2019 (COVID-19) is an acute infectious disease caused by the novel coronavirus (SARS-CoV-2) identified in 2019. The COVID-19 outbreak continues to have devastating consequences for human lives and the global economy. The B-LiFe mobile laboratory in Piedmont, Italy, was deployed for the surveillance of COVID-19 cases by large-scale testing of first responders. The objective was to assess the seroconversion among the regional civil protection (CP), police, health care professionals, and volunteers. The secondary objective was to detect asymptomatic individuals within this cohort in the light of age, sex, and residence. In this paper, we report the results of serological testing performed by the B-LiFe mobile laboratory deployed from 10 June to 23 July 2020. The tests included whole blood finger-prick and serum sampling for detection of SARS-CoV-2 spike receptor-binding domain (S-RBD) antibodies. The prevalence of SARS-CoV-2 antibodies was approximately 5% (294/6013). The results of the finger-prick tests and serum sample analyses showed moderate agreement (kappa = 0.77). Furthermore, the detection rates of serum antibodies to the SARS-CoV-2 nucleocapsid protein (NP) and S-RBD among the seroconverted individuals were positively correlated (kappa = 0.60), at least at the IgG level. Seroprevalence studies based on serological testing for the S-RBD protein or SARS-CoV-2 NP antibodies are not sufficient for diagnosis but might help in screening the population to be vaccinated and in determining the duration of seroconversion.

Introduction : Lung cancer is the most frequent and the deadliest cancer in the world. In non-small cell lung cancer (NSCLC), which represents 85% of all lung cancers, up to 55% patients relapse following surgery alone or in combination with chemotherapy or radiotherapy. Tumor-infiltrating lymphocytes play a key role in the control of the malignancy and display different phenotypes whilst interacting with cancer cells. Hence, immunoglobulin-A (IgA)-producing cells have been described to have an immunosuppressive function, promoting tumor development and growth in hepatocarcinoma and prostate cancer. Due to the important role of IgA in the lung, we aimed to study the prognostic significance of IgA+ cell infiltration in the epithelial and stromal compartments of resected NSCLCs. [...]

Macrophages are not only derived from circulating blood monocytes or embryonic precursors but also expand by proliferation. The origin determines macrophage fate and functions in steady state and pathological conditions. Macrophages predominantly infiltrate fibre-induced mesothelioma tumors and contribute to cancer development. Here, we revealed their ontogeny by comparing the response to needle-like mesotheliomagenic carbon nanotubes (CNT-7) with tangled-like non-mesotheliomagenic CNT-T. In a rat peritoneal cavity model of mesothelioma, both CNT induced a rapid macrophage disappearance reaction (MDR) of MHCIIlow resident macrophages generating an empty niche available for macrophage repopulation. Macrophage depletion after mesotheliomagenic CNT-7 was followed by a substantial inflammatory reaction, and macrophage replenishment completed after 7 days. Thirty days after non-mesotheliomagenic CNT-T, macrophage repopulation was still incomplete and accompanied by a limited inflammatory reaction. Cell depletion experiments, flow cytometry and RNA-seq analysis demonstrated that, after mesotheliomagenic CNT-7 exposure, resident macrophages were mainly replaced by an influx of monocytes, which differentiated locally into MHCIIhigh inflammatory macrophages. In contrast, the low inflammatory response induced by CNT-T was associated by the accumulation of self-renewing MHCIIlow macrophages that initially derive from monocytes. In conclusion, the mesotheliomagenic response to CNT specifically relies on macrophage niche recolonization by monocyte-derived inflammatory macrophages. In contrast, the apparent homeostasis after non-mesotheliomagenic CNT treatment involves a macrophage regeneration by proliferation. Macrophage depletion and repopulation are thus decisive events characterizing the carcinogenic activity of particles and fibres.

Mutualistic associations between insects and heritable bacterial symbionts are ubiquitous in nature. The aphid symbiont Serratia symbiotica is a valuable candidate for studying the evolution of bacterial symbiosis in insects because it includes a wide diversity of strains that reflect the diverse relationships in which bacteria can be engaged with insects, from pathogenic interactions to obligate intracellular mutualism. The recent discovery of culturable strains, which are hypothesized to resemble the ancestors of intracellular strains, provide an opportunity to study the mechanisms underlying bacterial symbiosis in its early stages. In this study, we analyzed the genomes of three of these culturable strains that are pathogenic to aphid hosts, and performed comparative genomic analyses including mutualistic host-dependent strains. All three genomes are larger than those of the host-restricted S. symbiotica strains described so far, and show significant enrichment in pseudogenes and mobile elements, suggesting that these three pathogenic strains are in the early stages of the adaptation to their host. Compared to their intracellular mutualistic relatives, the three strains harbor a greater diversity of genes coding for virulence factors and metabolic pathways, suggesting that they are likely adapted to infect new hosts and are a potential source of metabolic innovation for insects. The presence in their genomes of secondary metabolism gene clusters associated with the production of antimicrobial compounds and phytotoxins supports the hypothesis that S. symbiotia symbionts evolved from plant-associated strains and that plants may serve as intermediate hosts. Mutualistic associations between insects and bacteria are the result of independent transitions to endosymbiosis initiated by the acquisition of environmental progenitors. In this context, the genomes of free-living S. symbiotica strains provide a rare opportunity to study the inventory of genes held by bacterial

On Friday, 19 March 2021, WHO organized a virtual global workshop highlighting the need for a globally coordinated plan to increase SARS-CoV-2 genetic sequencing capacities to detect SARS-CoV-2 mutations and variants, and to monitor virus genomic evolution worldwide. One week later, in another virtual meeting, it focused on sero epidemiology for SARS-CoV-2 variants of concern and variants of interest. Efficient monitoring of the virus relies on the storage, handling and sharing of the genomic data and the associated metadata. In this manuscript, we demonstrate how the Bioconductor VariantExperiment class addresses these needs, offering a robust and efficient solution to the requirements laid out by the WHO.

Multigene panels are routinely used to assess for predisposing germline mutations in families at high breast cancer risk. The number of variants of unknown significance thereby identified increases with the number of sequenced genes. We aimed to determine whether tumor sequencing can help refine the analysis of germline variants based on second somatic genetic events in the same gene. Whole-exome sequencing (WES) was performed on whole blood DNA from 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53, or CHEK2 mutation. Rare variants were retained in a list of 735 genes. WES was performed on matched tumor DNA to identify somatic second hits (copy number alterations (CNAs) or mutations) in the same genes. Distinct methods (among which immunohistochemistry, mutational signatures, homologous recombination deficiency, and tumor mutation burden analyses) were used to further study the role of the variants in tumor development, as appropriate. Sixty-eight patients (97%) carried at least one germline variant (4.7 ± 2.0 variants per patient). Of the 329 variants, 55 (17%) presented a second hit in paired tumor tissue. Of these, 53 were CNAs, resulting in tumor enrichment (28 variants) or depletion (25 variants) of the germline variant. Eleven patients received variant disclosure, with clinical measures for five of them. Seven variants in breast cancer-predisposing genes were considered not implicated in oncogenesis. One patient presented significant tumor enrichment of a germline variant in the oncogene ERBB2, in vitro expression of which caused downstream signaling pathway activation. Tumor sequencing is a powerful approach to refine variant interpretation in cancer-predisposing genes in high-risk breast cancer patients. In this series, the strategy provided clinically relevant information for 11 out of 70 patients (16%), adapted to the considered gene and the familial clinical phenotype.

PURPOSE: Housekeeping genes (HKGs), reference or endogenous control genes, are vital to normalize mRNA levels between different samples. Since using inappropriate HKGs can lead to unreliable results, selecting the proper ones is critical for gene expression studies. To this end, normal human ovaries, as well as those from patients diagnosed with ovarian endometrioid adenocarcinoma (OEA), ovarian mucinous adenocarcinoma (OMA), ovarian serous papillary carcinoma (OSPC), and polycystic ovary syndrome (PCOS), were used to identify the most suitable housekeeping genes. METHODS: RNA was isolated from 5 normal human ovaries (52-79 years of age), 9 cancerous ovaries (3 OEA, 3 OMA, 3 OSPC; 49-75 years of age), and 4 PCOS ovaries (18-35 years of age) in women undergoing hysterectomy. cDNA was synthesized using a whole transcriptome kit, and quantitative real-time PCR was performed using TaqMan array 96-well plates containing 32 human endogenous controls in triplicate. RESULTS: Among 32 HKGs studied, RPS17, RPL37A, PPIA, 18srRNA, B2M, RPLP0, RPLP30, HPRT1, POP4, CDKN1B, and ELF1 were selected as the best reference genes. CONCLUSIONS: This study confirms recent investigations demonstrating that conventional HKGs, such as GAPDH and beta-actin, are not suitable reference genes for specific pathological conditions, emphasizing the importance of determining the best HKGs on a case-by-case basis and according to tissue type. Our results have identified reliable HKGs for studies of normal human ovaries and those affected by OEA, OMA, OSPC, or PCOS, as well as combined studies of control subjects vs. each cancer or PCOS group.

STUDY QUESTION: Can human theca cells (TCs) be differentiated in vitro? SUMMARY ANSWER: It is possible to differentiate human TCs in vitro using a medium supplemented with growth factors and hormones. WHAT IS KNOWN ALREADY: There are very few studies on the origin of TCs in mammalian ovaries. Precursor TCs have been described in neonatal mice ovaries, which can differentiate into TCs under the influence of factors from oocytes and granulosa cells (GCs). On the other hand, studies in large animal models have reported that stromal cells (SCs) isolated from the cortical ovarian layer can also differentiate into TCs. STUDY DESIGN, SIZE, DURATION: After obtaining informed consent, ovarian biopsies were taken from eight menopausal women (53-74 years of age) undergoing laparoscopic surgery for gynecologic disease not related to the ovaries. SCs were isolated from the ovarian cortex and in vitro cultured for 8 days in basic medium (BM) (G1), enriched with growth factors, FSH and LH in plastic (G2) or collagen substrate without (G3) or with (G4) a GC line. PARTICIPANTS/MATERIALS, SETTING, METHODS: To confirm TC differentiation, relative mRNA levels for LH receptor (Lhr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), cytochrome P450 17A1 (Cyp17a1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 2 (Hsd3b2) were assessed. Immunohistochemistry was also performed for their protein detection and a specific marker was identified for TCs (aminopeptidase-N, CD13), as were markers for theca and small luteal cells (dipeptidyl peptidase IV (CD26) and Notch homolog 1, translocation-associated (NOTCH1)). Finally, we analyzed cell ultrastructure before (Day 0) and after in vitro culture (Day 8), and dehydroepiandrosterone (DHEA) and progesterone levels in the medium using transmission electron microscopy (TEM) and ELISA, re

1.1. Purpose: As a result of increased life expectancy, the proportion of elderly patients with head and neck cancer is constantly rising. Transoral robotic surgery has been developed over the last ten years as a minimal invasive surgical procedure. The purpose of this study is to evaluate the place of this technique for elderly patients. 1.2. Methods & material: Study data related to elderly (age over 75 years) patients who underwent TORS between March 2008 and March 2018 were analyzed. 28 elderly patients were included; the different locations were 18 laryngeal (10 glottic and 8 supraglottic), 3 hypopharyngeal, 2 oral cavities and 5 oropharyngeal carcinoma respectively. 1.3. Results: 28 patients, 23 men and 5 women, with an average of 79 years old were successfully operated without external conversion. The 3-year Disease-Specific Survival (DSS) rate is 87.3% and the 3-year overall survival (OS) rate is 65,6%. Surgery was completed in a mean of 131 minutes (including exposure). All patients were extubated the same day (56%) or the day after the surgery (44%). Except for total laryngectomies, three patients (10.7%) received transient tracheostomies. Oral feeding was started after an average of 11 days after surgery. The hospitalization stay was 27 days on average. 1.4. Conclusions: Trans-oral Robotic surgery is a valuable technical option to address selected head and neck carcinoma in the elderly population. Early postoperative rehabilitation limits swallowing disorders and pulmonary complications. The surgical time is reduced compared to conventional open surgery which is a great advantage for this fragile population.

BACKGROUND & AIM: Chronic liver diseases are characterized by expansion of the small immature cholangiocytes - a mechanism named ductular reaction (DR) - which have the capacity to differentiate into hepatocytes. We investigated the kinetics of this differentiation, as well as analyzing several important features of the newly formed hepatocytes, such as functional maturity, clonal expansion and resistance to stress in mice with long-term liver damage. METHODS: We tracked cholangiocytes using osteopontin-iCreERT2 and hepatocytes with AAV8-TBG-Cre. Mice received carbon tetrachloride (CCl4) for >24 weeks to induce chronic liver injury. Livers were collected for the analysis of reporter proteins, cell proliferation and death, DNA damage, and nuclear ploidy; hepatocytes were also isolated for RNA sequencing. RESULTS: During liver injury we observed a transient DR and the differentiation of DR cells into hepatocytes as clones that expanded to occupy 12% of the liver parenchyma by week 8. By lineage tracing, we confirmed that these new hepatocytes derived from cholangiocytes but not from native hepatocytes. They had all the features of mature functional hepatocytes. In contrast to the exhausted native hepatocytes, these newly formed hepatocytes had higher proliferative capability, less apoptosis, a lower proportion of highly polyploid nuclei and were better at eliminating DNA damage. CONCLUSIONS: In chronic liver injury, DR cells differentiate into stress-resistant hepatocytes that repopulate the liver. The process might account for the observed parenchymal reconstitution in livers of patients with advanced-stage hepatitis and could be a target for regenerative purposes. LAY SUMMARY: During chronic liver disease, while native hepatocytes are exhausted and genetically unstable, a subset of cholangiocytes clonally expand to differentiate into young, functional and robust hepatocytes. This cholangiocyte cell population is a promising target for regenerative therapies in patie

Breast cancer is a heterogeneous disease for which the existence of monogenic and polygenic models of inheritance have been described for decades. Multigene panel testing is useful for the diagnosis of monogenic breast cancer predisposition, allowing to simultaneously sequence a large number of genes of several patients. However, this approach increases the identification of variants of unknown significance (VUS) that cannot be used in clinical decision-making. We first performed a systematic review and meta-analysis of studies conducting multigene panel testing on germline DNA of women with familial breast cancer. We found that using current panels, the probability of detecting a VUS is significantly higher than the probability of detecting a pathogenic variant. BRCA1 and BRCA2, the two most extensively studied breast cancer predisposing genes until now, were the only genes presenting the opposite trend. Reclassification of VUS in the other more recently identified genes is therefore required to drastically reduce the number of families for which genetic counselors are not able to draw a conclusion. Using a cohort of 70 unrelated breast cancer patients referred for genetic testing and without a BRCA1, BRCA2, TP53 or CHEK2 mutation, we demonstrated that sequencing the matching tumor can help reclassify germline VUS based on second events hitting the same gene and provide clinically relevant information. We then explored the concept of oligogenic inheritance of familial breast cancer, and obtained data suggestive of the pertinence of this model. Analyzing the combined segregation patterns of low-frequency variants in affected familial relatives of 54 of our index patients, we found significantly higher segregation rates of rare variants in cancer-related genes in families in which affected members share a similar breast cancer phenotype, as compared to phenotypically heterogeneous families. Furthermore, cancer genes with disease-cosegregating variants harbored second, (MED - Sciences médicales) -- UCL, 2019

BACKGROUND: When a patient is seen with a newly diagnosed oropharyngeal squamous cell carcinoma, it remains unclear to the treating physicians how fast the tumor growth rate is. METHODS: From patients with oropharynx squamous cell carcinoma treated by radiotherapy, the investigators selected comparable diagnostic CT-scan (DiCT) and radiotherapy planning CT-scan (RtCT). Tumor and pathological lymph node volumes were measured in order to calculate tumor progression. RESULTS: From the selection of 19 patients, the mean absolute tumor progression rate was 0.23 ± 0.2 cm3 /d and mean relative progression rate was 1.84 ± 1.64%/d. Mean tumor doubling time is 286 days (range 7-1282 days), demonstrating a wide range of tumor growth pattern. Significant tumor progression (>20%) between DiCT and RtCT was shown in 73% of patients, and 53% of the patients were seen a tumor progression of >50% within a mean waiting time of 42.1 days. Kaplan-Meier curves showed a non-significative link between fast progression tumors (>1%/d) and higher risk of recurrence (HR: 2.2; P = .23). CONCLUSIONS: Tumor progression can be assessed based on DiCT and RtCT. Treatment delay should be avoided at all cost. Different growth patterns were evidenced. For the fast-growing tumors subgroup, pejorative clinical outcomes were suggested. Prospective studies are needed to confirm a link between fast-growing tumors and higher risk for recurrence.

BACKGROUND. Cell transplantation is in clinical development for the treatment of various ailments including acquired and inborn hepatic diseases. Detection and quantification of the donor cells after infusion remain difficult. Traditional methods (sex-based FISH, HLA mismatch, and Short Tandem Repeat PCR) can only achieve low levels of sensitivity (1%) and therefore are seldom used. The use of a droplet digital PCR (ddPCR) assay based on mismatch of null alleles is a promising alternative. METHODS. We selected genes with a high frequency of null genotype in the general population (SRY, RHD, TRY6, LEC3C, GSTM1, and GSTT1) and investigated their expression by liver progenitor cell donors and liver cell therapy recipients, in order to identify genes of interest for each donor/recipient couple. We first validated the detection of microchimerism by ddPCR and then used these assays to detect and quantify microchimerism in pre- and postinfusion liver biopsies. RESULTS. We validated the ddPCR detection of the selected genes based on linearity, precision, lack of inhibition, and accuracy, and we established limits of blank, limits of detection, and limits of quantification to ensure the reliability of the results. After genotyping donors and recipients, we were able to identify at least one gene of interest for each donor/recipient couple. We detected donor cells in the three patients posttransplantation. However, analysis of several biopsies taken at the same timepoint revealed a heterogeneous cell distribution. In addition, the values obtained remained below the limit of quantification. Therefore, the actual quantification of microchimerism may not be entirely accurate. CONCLUSIONS Overall, our study demonstrates that the detection of microchimerism post-liver cell transplantation can be performed using ddPCR amplification of null allele genes expressed by the donor but absent from the recipient. However, this technique can be extended to other cell types and target organs

Four isothermal recombinase polymerase amplification assays (RPA) were developed for fast in-field identification of RPAs targeted three specific sequences (i.e. BA_5345 chromosomal marker, lethal factor -pXO1, and capsule biosynthesis -pXO2), and a conserved sequence of the group in the adenylate cyclase gene (). -specific RPA assays were first tested on purified genomic DNAs (n=60) containing 11 representatives of , then on soil (n=8) and white powder (n=8) samples spiked with inactivated spores and/or other biological agents. The RPA assays were also tested in another laboratory facility which blindly provided DNA and lysate samples (n=30, including 20 strains). RPA tests displayed 100% specificity and sensitivity. The hands-off turnaround time at 42°C ranged from 5 to 6 min for 10 genomic copies. The analytical sensitivity of each RPA was ∼10 molecules per reaction. Besides, BA_5345 and RPA assays were assessed in field conditions on a series of surface swabs (n=13 with 11 swabs contaminated with spores) and blindly dispatched to the field laboratory by a CBRN sampling team. None of the 13 samples, expect the control, tested positive for and all samples harvested on spores-contaminated surfaces tested positive with -RPA assay. All three -specific RPA assays proved suitable for rapid and reliable identification of and could therefore easily be used by first responders in field conditions to quickly discriminate between a deliberate release of spores and a hoax attack involving "white-powders". In recent decades, particularly following 9/11 and the Amerithrax attack, the world experienced several attempts to sow panic and chaos in the society through thousands of white-powder copycats using household powders to mimic a real bioterrorism attack. In such circumstances, field deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test them directly using a fast and reliable assay for detecting the presence of Wh

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN SIZE, AND DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in-vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical fi

While in mice various studies have described the completion of spermatogenesis in vitro using either organotypic culture of prepubertal testicular tissue or 3D culture of isolated cells, in humans it has not been possible to achieve germ cell differentiation from immature testicular tissue (ITT). In our study, we evaluated the ability of human ITT to differentiate via a long-term organotypic culture of frozen–thawed 1 mm3 testicular fragments from five prepubertal boys in two different culture media. Tissue and supernatants were analyzed at regular intervals up to day 139. Sertoli cell (SC) viability and maturation was evaluated using immunohistochemistry (IHC) for SOX9, GDNF, anti-Mullerian hormone (AMH) and androgen receptor (AR), and AMH concentration in supernatants. Spermatogonia (SG) and proliferating cells were identified by MAGE-A4 (for SG) and Ki67 (for proliferating cells) via immunohistochemistry (IHC). Apoptotic cells were studied by active caspase 3. To evaluate Leydig cell (LC) functionality testosterone was measured in the supernatants and steroidogenic acute regulatory protein (STAR) IHC was performed. Germ cell differentiation was evaluated on Hematoxylin-Eosin histological sections, via IHC for synaptonemal complex 3 (SYCP3) for spermatocytes, Protein boule-like (BOLL) for spermatocytes and round spermatids, angiotensin-converting enzyme (ACE), protamine 2 and transition protein 1 (for elongated spermatids) and via chromogenic in situ hybridization (CISH). We reported the generation of meiotic and postmeiotic cells after 16 days of culture, as shown by the histological analyses, the presence of differentiation markers and the increase of haploid germ cells. We showed SC viability and maturation by a decrease of AMH secretion in the supernatants (p ≤ 0.001) while the number of SOX9 positive cells did not show any variation. A decrease of spermatogonia (p ≤ 0.001) was observed. The number of apoptotic cells did not vary. LC functionality was shown by

RESEARCH QUESTION: Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? DESIGN: Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. RESULTS: The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. CONCLUSIONS: This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.

RESEARCH QUESTION: Does ulipristal acetate (UPA) modify the expression of genes related to apoptosis or the extracellular matrix in uterine myomas and are any modifications associated with a clinical response? DESIGN: Targeted analysis of 176 apoptosis- or extracellular-matrix-related genes was conducted using polymerase chain reaction (PCR) arrays. Relevant results were validated by quantitative PCR. Four groups were established: responsive short-term (one course, n = 9), responsive long-term (two to four courses, n = 9), non-responsive (n = 9), and the control group who was not given any hormone therapy (n = 9). The clinical response was monitored by medical imagery and considered significant when volume reduction was greater than 25%. RESULTS: Compared with untreated myomas, significant changes in expression of four genes were found in UPA-treated myomas. Gene expression of integrin subunit beta 4 was repressed by UPA treatment (fold change [FC] = -12.50, P < 0.001, q < 0.001), tenascin-C expression was downregulated in UPA-responsive patients (FC = -2.50, P = 0.010, q = 0.090), survivin was repressed in short-term UPA-responsive tumours (FC = -7.69, P < 0.001, q = 0.010), and catenin delta 2 gene expression was upregulated in non-responsive myomas (FC = +7.36, P < 0.001, q = 0.010). CONCLUSION: This characterization provides the first molecular distinction between myomas responsive or non-responsive to UPA treatment.

STUDY QUESTION: How does the formation of the blood-testis barrier (BTB), as reflected by the expression of connexin 43 and claudin 11 proteins during the pubertal transition period, take place in vitro compared to samples from a large cohort of pre/peripubertal boys? SUMMARY ANSWER: The BTB connexin 43 and claudin 11 expression patterns appeared to be partially achieved in organotypic culture when compared to that in samples from 71 pre/peripubertal patients. WHAT IS KNOWN ALREADY: Although alterations in the protein expression patterns of the BTB, whose main components are connexin 43 and claudin 11, are known to be associated with impaired spermatogenesis in mice and adult men, there is a lack of knowledge on its formation in pre-peripubertal human tissue both in vitro and in vivo. Moreover, despite Sertoli cell (SC) maturation during long-term organotypic culture of immature testicular tissue (ITT), initiation of spermatogenesis has not yet been achieved. STUDY DESIGN SIZE, AND DURATION: Histological sections from 71 pre-peripubertal patients were evaluated for the formation of the BTB acting as in-vivo controls according to age, SC maturation, clinical signs of puberty and germ cell differentiation. Testicular tissue fragments retrieved from three prepubertal boys were cultured in a long term organotypic system to analyze the BTB formation and expression pattern in correlation with SC maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testicular histological sections from 71 patients aged 0-16 years who underwent a biopsy between 2005 and 2014 to preserve their fertility before gonadotoxic treatment were examined. Immunohistochemistry (IHC) results for connexin 43 and claudin 11 as BTB markers, using a semi-quantitative score for their expression, and for Anti-Mullerian hormone (AMH), as SC maturation marker, were analyzed. Germ cell differentiation was evaluated on Hematoxylin-Eosin sections. Tanner stages at the time of biopsy were recorded from medical fi

[Research question] Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? [Design] Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. [Results] The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. [Conclusions] This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.

Many genetic risk factors have been identified for causing venous thromboembolism (VTE). Most of them affect the function of natural anticoagulant pathways, particularly the protein C system, although recent studies suggest a role of components of the hematopoietic pathway in the etiology of venous thrombosis. In this case-control study, we aimed to determine the frequency of prothrombin G20210A and factor V Leiden (FVL) G1691A polymorphisms and protein C, protein S, and antithrombin III deficiencies in the East Algerian population and to investigate whether these genetic factors are associated with VTE. On the other hand, our study tends to evaluate the status of JAK2V617F and calreticulin (CALR) mutations among these cases. The participants consisted of 121 cases with VTE and 146 healthy controls. Polymorphisms of FVL G1691A and prothrombin G20210A were genotyped by polymerase chain reaction (PCR) restriction fragment length polymorphism. JAK2-V617F and calreticulin mutations were analyzed by quantitative PCR and PCR followed by capillary electrophoresis sequencing, respectively. Protein C, protein S, and antithrombin levels were determined and then hereditary deficiencies were identified. Of all cases and controls, none was a carrier of the antithrombin III deficiency, prothrombin gene G20210A, and CALR mutations. Only 1 case reported having a positive JAK2 mutation (mutant allele burden was 15%). The FVL mutation (GA/AA) was found in 14 (11.6%) cases and 2 (1.4%) controls and it was significantly different between both the groups (P = .001). Deficiencies of protein S and protein C were detected in 17 (18.8%) cases. The univariate analysis resulted in a significant impact of FVL (odds ratio [OR] = 9.4, 95% confidence interval [CI] = 2.1-42.3; P = .003) and of protein S deficiency (OR = 16.9, 95% CI =2.1-132.8, P = .007) on the VTE status. Both factors stayed significant after adjustment for sex and age. The OR of the protein C deficiency was slightly elevated (OR

Background and Aims: Alagille syndrome (AS) is an autosomal dominant multi-systemic disorder caused by pathogenic variants in JAG1 and NOTCH2. Characteristic findings include hepatic involvement with bile duct paucity and 20-50% eventually need a liver transplantation. Post-LT Tacrolimus induced nephropathy is well recognised and 40% of AS patients have an underlying renal anomaly. In the current study we analysed the impact of LT and Tacrolimus on the evolution of renal function (RF) in children with AS. Methods: Retrospective study including 50 children that satisfied 3 of 5 major Alagille syndrome criteria and under regular follow-up at our centre between 1984 and 2016. Clinical, biochemical and radiological data were collected at similar time points of follow-up among the transplanted and non-transplanted children. The time points were at diagnosis or at LT and after 1-2 years, 2-3 years, 3-5 years, 5-7 years and 7-10 years of follow-up. The RF was estimated by glomerular filtration rate (eGFR) using the updated Schwartz formula. The RF outcomes of children with AS having undergone LT were compared with those without LT and also with children having undergone LT for non-AS related indication but without associated nephropathy. Results: 28 of 50 (56%) included AS children underwent LT and were compared with 77 children transplanted for non-AS indications. Mean eGFR post-LT in AS patients and non-AS patients were 93.8 mL/min and 143.2 mL/min, respectively (difference: 49.4 mL/min, p<0.0001). Among children with AS mean eGFR observed in those who did not receive LT was 87.9 mL/min, -5.9 mL/min compared to those who received LT though this was statistically insignificant (p=0.32). Presence of renal ultrasound abnormalities was correlated to RF impairment in AS patients, with or without LT: -14.6 mL/min (98.5 mL/min vs 83.9 mL/min, p=0.03) and -40.9 mL/min (97.8 mL/min vs 56.9 mL/min, p<0.0001), respectively. Conclusions: Post-LT renal function outcomes are significa

Background and Aims: Alagille syndrome (AS) is an autosomal dominant multi-systemic disorder caused by pathogenic variants in JAG1 and NOTCH2. Characteristic findings include hepatic involvement with bile duct paucity and 20-50% eventually need a liver transplantation. Post-LT Tacrolimus induced nephropathy is well recognised and 40% of AS patients have an underlying renal anomaly. In the current study we analysed the impact of LT and Tacrolimus on the evolution of renal function (RF) in children with AS. Methods: Retrospective study including 50 children that satisfied 3 of 5 major Alagille syndrome criteria and under regular follow-up at our centre between 1984 and 2016. Clinical, biochemical and radiological data were collected at similar time points of follow-up among the transplanted and non-transplanted children. The time points were at diagnosis or at LT and after 1-2 years, 2-3 years, 3-5 years, 5-7 years and 7-10 years of follow-up. The RF was estimated by glomerular filtration rate (eGFR) using the updated Schwartz formula. The RF outcomes of children with AS having undergone LT were compared with those without LT and also with children having undergone LT for non-AS related indication but without associated nephropathy. Results: 28 of 50 (56%) included AS children underwent LT and were compared with 77 children transplanted for non-AS indications. Mean eGFR post-LT in AS patients and non-AS patients were 93.8 mL/min and 143.2 mL/min, respectively (difference: 49.4 mL/min, p<0.0001). Among children with AS mean eGFR observed in those who did not receive LT was 87.9 mL/min, -5.9 mL/min compared to those who received LT though this was statistically insignificant (p=0.32). Presence of renal ultrasound abnormalities was correlated to RF impairment in AS patients, with or without LT: -14.6 mL/min (98.5 mL/min vs 83.9 mL/min, p=0.03) and -40.9 mL/min (97.8 mL/min vs 56.9 mL/min, p<0.0001), respectively. Conclusions: Post-LT renal function outcomes are significa

OBJECTIVE: Salvage surgery in recurrent SCCHN is associated with poor outcomes. This study aimed to better identify suitable surgical candidates and those at high risk of new recurrence. MATERIALS AND METHODS: Single-center retrospective analysis of 109 patients undergoing salvage surgery for recurrent SCCHN. Univariate and multivariate analyses were used to identify prognostic factors affecting disease-free survival (DFS). RESULTS: The following factors showed a significant impact on DFS: Disease-free interval >6months [HR 0.53; p=0.04], age>70years [HR 0.26; p=0.03], primary chemoradiotherapy [HR 2.39; p<0.01] compared to radiotherapy, oropharynx [HR 5.46; p<0.01] and hypopharynx [HR 3.92; p=<0.01] sites, compared to larynx, initial stage III [HR 7.10; p<0.01] and stage IV [HR 4.13; p<0.01], compared to stage I, locoregional recurrence [HR 4.57; p<0.01], compared to local recurrence. Univariate analysis also identified significant postoperative predictors of poor DFS including flap reconstruction [HR 3.44; p<0.01], postoperative complications [HR 2.09; p=0.01], positive margins [HR 3.64; p<0.01] and close margins [HR 3.83; p<0.01]. On multivariate analysis, oropharynx site [HR 3.98; p<0.01], initial stage III [HR 5.93; p<0.01] and locoregional recurrence [HR 2.93; p=0.04] were independent preoperative prognostic factors for DFS. Positive margins [HR 2.32; p=0.04], close margins [HR 2.94; p=0.02], extracapsular spread (ECS) [HR 4.04; p=0.03] and postoperative complications [HR 3.64; p<0.01] were independent postoperative prognostic factors. CONCLUSIONS: Patients with advanced primary nonlaryngeal tumor and locoregional recurrence have limited success with salvage surgery. Because patients with positive margins and ECS are at high risk of relapse, adjuvant treatment should be discussed.

Objectives and study: Alagille syndrome (AS) is an autosomal dominant multi-systemic disorder caused by pathogenic variants in JAG1 and NOTCH2. Characteristic findings include hepatic involvement with bile duct paucity and 20-50% eventually need a liver transplantation. Post-LT Tacrolimus induced nephropathy is well recognised and 40% of AS patients have an underlying renal anomaly. In the current study we analysed the impact of LT and Tacrolimus on the evolution of renal function (RF) in children with AS. Methods: Retrospective study including 50 children that satisfied 3 of 5 major Alagille syndrome criteria and under regular follow-up at our centre between 1984 and 2016. Clinical, biochemical and radiological data were collected at similar time points of follow-up among the transplanted and non-transplanted children. The time points were at diagnosis or at LT and after 1-2 years, 2-3 years, 3-5 years, 5-7 years and 7-10 years of follow-up. The RF was estimated by glomerular filtration rate (eGFR) using the updated Schwartz formula. The RF outcomes of children with AS having undergone LT were compared with those without LT and also with children having undergone LT for non-AS related indication but without associated nephropathy. Results: 28 of 50 (56%) included AS children underwent LT and were compared with 77 children transplanted for non-AS indications. Mean eGFR post-LT in AS patients and non-AS patients were 93.8 mL/min and 143.2 mL/min, respectively (difference: 49.4 mL/min, p<0.0001). Among children with AS mean eGFR observed in those who did not receive LT was 87.9 mL/min, -5.9 mL/min compared to those who received LT though this was statistically insignificant (p=0.32). Presence of renal ultrasound abnormalities was correlated to RF impairment in AS patients, with or without LT: -14.6 mL/min (98.5 mL/min vs 83.9 mL/min, p=0.03) and -40.9 mL/min (97.8 mL/min vs 56.9 mL/min, p<0.0001), respectively. Conclusion: Post-LT renal function outcomes are significa

BACKGROUND AND AIMS Ductular reaction (DR) is identified by using CK7 staining and respresents a ductular phenotype, possibly arising from proliferation of cholangiocytes and progenitor cells. DR has been linked to hepatic fibrosis progressing in HCV and hemochromatosis. It is frequently seen on protocol biopsies (PB) post-liver transplant (LT) and given its origin it’s unclear if its linked to fibrogenesis or hepatocyte regeration after an insult. This was analyzed in the current study using paired PB’s to test if CK7 quantification on the baseline PB could predict the course of fibrogenesis in the follow-up PB. METHODS Children who underwent LT from 2012 to 2014 and had <2 PB at an interval of 1-2 years between each other were included. The first PB was labeled as “baseline biopsy” and the successive biopsy as “follow-up biopsy”. The change of fibrosis severity between the “baseline” and “follow-up” biopsy was termed as “prospective change in fibrosis”. Each biopsy was evaluated for inflammation and fibrosis using the Metavir and LAFSc system. The baseline biopsy was stained with CK7 and digitally evaluated to abtain “CK7-positive area percentage” i.e. CK7-stained area expressed as percentage of the total biopsy area. The association between CK7-positive area percentage on baseline biopsy and “prospective change in fibrosis” scores, ductular reaction, lobular inflammation, and portal tract inflammation was assessed using ordinal logistic regression models. RESULTS Our study included 64 pared PB from 32 children. The baseline PB stained for CK7, were taken at a mean of 2.89 years post-LT. The time interval between the two paired PB’s was 1.41 years. CK7-positive area percentage was significantly associated with baseline fibrosis, extent of ductular reaction (p=.006) and portal tract inflammation (p=.01). There was a no significant association between the CK7-positive area percentage of the baseline biopsy and the “prospective change in fibrosis” as assessed by the

BACKGROUND AND AIMS Liver allograft fibrosis is often seen on protocol liver biopsies and class II donor specific HLA antibodies have been implicated. Evidence lacks to determine appropriate MFI cut off values or on relative importance of subtypes. METHODS Cross-sectional study including stable LT recipient children having undergone protocol biopsy between 2012-2015. Biopsies were assessed for fibrosis and inflammation, HLA antibody detection using Luminex platform wad done pre-LT and simultaneous to protocol biopsy. Date evaluation and statistics: Impact of HLA antibodies and other variables was analyzed using cumulative logistic regression. Then Polyserial correlation coefficients were computed for MFI of antibody subclasses and histological characteristics and Receiver Operating Characteristic curve used to determine MFI threshold. RESULTS 102 children were included, allograft fibrosis and portal fibrosis correlated to presence of post-LT class II DSA (OR=6.13, p=0.01 and OR=5.18, p=0.02 respectively). Allograft inflammation significantly correlated to presence of post-LT class II DSA in the portal area (OR=8.02,p<0.01). Higher polyserial correlation coefficients with portal inflammation and fibrosis were found for DQ (PCC=0.77 and 0.50) than for DP or DR antibody subclasses. The DQ subclass enabled 5000 MFI enabled a sensitivity (83.3% for inflammation and 75.0% for fibrosis) and specificity (71.4% for inflammation and 57.1% for fibrosis). CONCLUSIONS Among the post-LT class II DSa, DQ subclass with MFI>5000 is associated with allograft inflammation and fibrosis in the portal area.

Background and Aims: Ductular reaction (DR) is identified by using CK7 staining and represents a ductular phenotype, possibly arising from proliferation of cholangiocytes and progenitor cells. DR has been linked to hepatic fibrosis progression in HCV and hemochromatosis. It is frequently seen on protocol biopsies (PB) post-liver transplant (LT) and given its origin it’s unclear if its linked to fibrogenesis or hepatocyte regeneration after an insult. This was analyzed in the current study using paired PB’s to test if CK7 quantification on the baseline PB could predict the course of fibrogenesis in the follow up PB. Methods: Children who underwent LT from 2012 to 2014 and had ≥2 PB at an interval of 1-2 years between each other were included. The first PB was labeled as “baseline biopsy” and the successive biopsy as “follow-up biopsy.” The change of fibrosis severity between the “baseline” and “follow-up” biopsy was termed as “prospective change in fibrosis.” Each biopsy was evaluated for inflammation and fibrosis using the Metavir and LAFSc system. The baseline biopsy was stained with CK7 and digitally evaluated to obtain “CK7 – positive area percentage” i.e. CK7-stained area expressed as percentage of the total biopsy area. The association between CK7-positive area percentage on baseline biopsy and “prospective change in fibrosis” scores, ductular reaction, lobular inflammation, and portal tract inflammation was assessed using ordinal logistic regression models. Results: Our study included 64 pared PB from 32 children. The baseline PB stained for CK7, were taken at a mean of 2.89 years post-LT. The time interval between the two paired PB’s was 1.41 years. CK7-positive area percentage was significantly associated with baseline fibrosis, extent of ductular reaction (p = 0.006) and portal tract inflammation (p = 0.01). There was a no significant association between the CK7-positive area percentage of the baseline biopsy and the “prospective change in fibrosis” as asse

BACKGROUND AND AIMS Liver allograft fibrosis is often seen on protocol liver biopsies and class II donor specific HLA antibodies have been implicated. Evidence lacks to determine appropriate MFI cut off values or on relative importance of subtypes. METHODS Cross-sectional study including stable LT recipient children having undergone protocol biopsy between 2012-2015. Biopsies were assessed for fibrosis and inflammation, HLA antibody detection using Luminex platform wad done pre-LT and simultaneous to protocol biopsy. Date evaluation and statistics: Impact of HLA antibodies and other variables was analyzed using cumulative logistic regression. Then Polyserial correlation coefficients were computed for MFI of antibody subclasses and histological characteristics and Receiver Operating Characteristic curve used to determine MFI threshold. RESULTS 102 children were included, allograft fibrosis and portal fibrosis correlated to presence of post-LT class II DSA (OR=6.13, p=0.01 and OR=5.18, p=0.02 respectively). Allograft inflammation significantly correlated to presence of post-LT class II DSA in the portal area (OR=8.02,p<0.01). Higher polyserial correlation coefficients with portal inflammation and fibrosis were found for DQ (PCC=0.77 and 0.50) than for DP or DR antibody subclasses. The DQ subclass enabled 5000 MFI enabled a sensitivity (83.3% for inflammation and 75.0% for fibrosis) and specificity (71.4% for inflammation and 57.1% for fibrosis). CONCLUSIONS Among the post-LT class II DSa, DQ subclass with MFI>5000 is associated with allograft inflammation and fibrosis in the portal area.

Objectives and Study: ADHLSC (adult derived human liver stem/progenitor cells) infusions are undergoing clinical trials in treatment of unborn errors of metabolism. One objective is to increase the hepatic engraftment of the infuseed ADHLSC. Rolling and adhesion are essential steps for a succesful engraftment. Alternative methods are considered to avoid trypsinization, known to damage integrins during culture passages, and subsequently adhesion. Selction ligands are in addition insufficiently expressed, which may be compensated by attachment of sialyl Lewis X to the celles. Methods: ADHLSC were cultured on thermosensitive polymer and harvested by non enzymatic dissociation solution, and their adhesion properties was compared bo that of control cells cultured on CellBind (Corning, Wiesbaden, Germany) and harvested by trypsinization. Adhesion was evaluated by shear stress test utilizing µ-Sllide I Luer (ibidi GmBH, Martinsried, Germany) coated with vascular cell adhesion protein 1 (VCAM-1) or collagen type I. Secondly, the effect of the selectin ligand sialyl Lewis X attached to ADHLSC was compared to control cells for the rolling capacity, using the shear stress test with µ-Slide I Luer coated with E-selectin. Results: ADHLSCs cultured on thermosensitive polymer and harvested by non enzymatic dissociation solution showed a 38% higher adhesion to VCAM-1 coated µ-Slide I Luer (465 cells/field versus 338 cells/field in controls, 95% CI (92.2, 161.3) (p<0.01,n=4)). No significant chang in adhesion potential was seen on collagen type I coated µ-Slide I Luer (573 cells/field versus 579 cells/field in controls). Attachment of sialyl Lewis X to ADHLSC led to consideerable increase of rolling to E-selectin coated µ-Slide I Luer (from 5 cells in controls to 289 cells/field (p<0.01, n=3). Conclusion: We demonstrate that ADHLSC cultured on thermosensitive polymer, harvested by non enzymatic dissociation solution and attachment of sialyl Lewis X significantly increase respectivel

Histological quantification of apha smooth muscle actin predicts future graft fibrosis in pediatric liver transplant recipients Varma S1, Stéphenne X1, Komuta M2, Bouzin.C3, Ambroise J4, Smets F1, Reding R5, and Sokal EM1 Université Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroentérologie et Hépatologie Pédiatrique and Pediatric Research Unit, 2Service de Anatomopathologie, 3 Imaging Platform (2IP) Institut de Recherche Expérimentale et Clinique (IREC) , 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC) 5 Unités de Chirurgie Pédiatrique, Brussels, Belgium Aims: To evaluate significance of alpha smooth muscle actin (ASMA) expression on liver biopsy as predictor of future graft fibrosis in pediatric liver transplant (LT) recipients. Background: : Activated hepatic stellate cells express cytoplasmic protein-alpha smooth muscle actin (ASMA) and subsequently secrete collagen leading to liver fibrosis. As activation of stellate cells precedes collagen deposition, we hypothesize that quantification of ASMA can predict the severity of future fibrosis. Patients: Stable pediatric LT recipients transplanted between 2006-20012, with two protocol biopsies less than two years apart and first being at more than 1year post LT. Patients with biliary, vascular complications, autoimmune hepatitis, hepatitis B or C infection, re-transplantation, or those with inadequate biopsy size were excluded. Methods: Metavir and liver allograft fibrosis scoring (LAFSc) used for fibrosis assessment. Automated staining for ASMA followed by digital quantification of ASMA positive area percentage was done. Fibrosis on initial biopsy specimen was labelled “current fibrosis” and on next biopsy labelled “prospective fibrosis”. The change between “current” and “prospective” fibrosis score was “prospective change in fibrosis”. Bile duct proliferation, lobular inflammation and portal tract infiltration was also evaluated.

Allograft inflammation and fibrosis among maintenance pediatric liver transplant recipients – genetic predisposition and antibodies, connecting the missing links. Varma S1, Latinne D2, Komuta M3, Ambroise J4, Smets F1, Baldin P3,Reding R5 , Stephenne X1, and Sokal EM1 Université Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroentérologie et Hépatologie Pédiatrique, 3Service de Anatomopathologie 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC), 5 Paediatric Surgery and Transplantation Unit, Brussels, Belgium Aim: To determine impact of HLA allo-antibodies, non-HLA auto-antibodies and HLA-DRB1 genotype amongst stable, long term pediatric liver transplantation (LT) recipients; on allograft health. Background: Role of HLA allo-antibodies and non-HLA auto-antibodies in late allograft fibrosis and inflammation is unclear in pediatric LT, though inferior long-term outcomes with class II donor specific HLA antibodies (Class-II DSA) post LT are suggested. HLA DRB1*03 or 04 genotype predisposes to auto-immune hepatitis (AIH), prototype of antibody related hepatic inflammation. Patients: Stable LT recipients transplanted in 2006-2012 and with >2 protocol biopsies were included. Autoimmune hepatitis, hepatitis B or C infection as indication of LT; ABOi graft, biliary or vascular complications post LT, re-transplantation were exclusion criteria. Methods Data were collected at 1-month pre-LT and simultaneous to last protocol biopsy. HLA, ANA, SMA, LKM antibodies, HLA-DRB1 genotype of recipients and histological aspects of biopsy, were collected. Histological parameters including rejection, bile duct proliferation, lobular inflammation, and portal tract infiltration were assessed while fibrosis was evaluated using Metavir and liver allograft fibrosis scoring (LAFSc). Results 15 of 89 included children had class-II DSA post LT. Class-II DSA was associated with allograft fibrosis using Metavir and

Allograft inflammation and fibrosis among maintenance pediatric liver transplant recipients – genetic predisposition and antibodies, connecting the missing links. Varma S1, Latinne D2, Komuta M3, Ambroise J4, Smets F1, Baldin P3,Reding R5 , Stephenne X1, and Sokal EM1 Université Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroentérologie et Hépatologie Pédiatrique, 3Service de Anatomopathologie 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC), 5 Paediatric Surgery and Transplantation Unit, Brussels, Belgium Aim: To determine impact of HLA allo-antibodies, non-HLA auto-antibodies and HLA-DRB1 genotype amongst stable, long term pediatric liver transplantation (LT) recipients; on allograft health. Background: Role of HLA allo-antibodies and non-HLA auto-antibodies in late allograft fibrosis and inflammation is unclear in pediatric LT, though inferior long-term outcomes with class II donor specific HLA antibodies (Class-II DSA) post LT are suggested. HLA DRB1*03 or 04 genotype predisposes to auto-immune hepatitis (AIH), prototype of antibody related hepatic inflammation. Patients: Stable LT recipients transplanted in 2006-2012 and with >2 protocol biopsies were included. Autoimmune hepatitis, hepatitis B or C infection as indication of LT; ABOi graft, biliary or vascular complications post LT, re-transplantation were exclusion criteria. Methods Data were collected at 1-month pre-LT and simultaneous to last protocol biopsy. HLA, ANA, SMA, LKM antibodies, HLA-DRB1 genotype of recipients and histological aspects of biopsy, were collected. Histological parameters including rejection, bile duct proliferation, lobular inflammation, and portal tract infiltration were assessed while fibrosis was evaluated using Metavir and liver allograft fibrosis scoring (LAFSc). Results 15 of 89 included children had class-II DSA post LT. Class-II DSA was associated with allograft fibrosis using Metavir and

Histological quantification of apha smooth muscle actin predicts future graft fibrosis in pediatric liver transplant recipients Varma S1, Stéphenne X1, Komuta M2, Bouzin.C3, Ambroise J4, Smets F1, Reding R5, and Sokal EM1 Université Catholique de Louvain, Cliniques Universitaires St Luc, 1Service de Gastroentérologie et Hépatologie Pédiatrique and Pediatric Research Unit, 2Service de Anatomopathologie, 3 Imaging Platform (2IP) Institut de Recherche Expérimentale et Clinique (IREC) , 4 Centre for Applied Molecular Technologies (CTMA), Institut de Recherche Expérimentale et Clinique (IREC) 5 Unités de Chirurgie Pédiatrique, Brussels, Belgium Aims: To evaluate significance of alpha smooth muscle actin (ASMA) expression on liver biopsy as predictor of future graft fibrosis in pediatric liver transplant (LT) recipients. Background: Activated hepatic stellate cells express cytoplasmic protein-alpha smooth muscle actin (ASMA) and subsequently secrete collagen leading to liver fibrosis. As activation of stellate cells precedes collagen deposition, we hypothesize that quantification of ASMA can predict the severity of future fibrosis. Patients: Stable pediatric LT recipients transplanted between 2006-20012, with two protocol biopsies less than two years apart and first being at more than 1year post LT. Patients with biliary, vascular complications, autoimmune hepatitis, hepatitis B or C infection, re-transplantation, or those with inadequate biopsy size were excluded. Methods: Metavir and liver allograft fibrosis scoring (LAFSc) used for fibrosis assessment. Automated staining for ASMA followed by digital quantification of ASMA positive area percentage was done. Fibrosis on initial biopsy specimen was labelled “current fibrosis” and on next biopsy labelled “prospective fibrosis”. The change between “current” and “prospective” fibrosis score was “prospective change in fibrosis”. Bile duct proliferation, lobular inflammation and portal tract infiltration was also evaluated. Re

AIMS: Slide digitalization has brought pathology to a new era, including powerful image analysis possibilities. However, while being a powerful prognostic tool, immunostaining automated analysis on digital images is still not implemented worldwide in routine clinical practice. METHODS AND RESULTS: Digitalized biopsy sections from two independent cohorts of patients, immunostained for membrane or nuclear markers, were quantified with two automated methods. The first was based on stained cell counting through tissue segmentation, while the second relied upon stained area proportion within tissue sections. Different steps of image preparation, such as automated tissue detection, folds exclusion and scanning magnification, were also assessed and validated. Quantification of either stained cells or the stained area was found to be correlated highly for all tested markers. Both methods were also correlated with visual scoring performed by a pathologist. For an equivalent reliability, quantification of the stained area is, however, faster and easier to fine-tune and is therefore more compatible with time constraints for prognosis. CONCLUSIONS: This work provides an incentive for the implementation of automated immunostaining analysis with a stained area method in routine laboratory practice.

To determine predisposing factors of idiopathic allograft fibrosis among pediatric liver transplant recipients.

Purpose: Heart failure (HF) is a complex syndrome. Its appropriate management should combine several health measurements. We assessed the relationship between the Kansas City Cardiomyopathy Questionnaire (KCCQ) and the Pocock’s clinical score. // Methods: We conducted a prospective registry of HF outpatients. The main outcome was occurrence of death or hospitalization during a 6-month follow-up. A multivariate logistic regression was performed, including the KCCQ overall summary score, the Pocock’s clinical score and their interaction in the model. // Results : From January 2008 to December 2010, 143 patients were involved. Mean age of patients was 68 years, and 74 % were men. KCCQ’s overall summary score and Pocock’s clinical score were inversely correlated (r = −0.24, p = 0.026). A total of 61 (42.7 %) events occurred. There was a high proportion of events (77.8 %) in patients with a Pocock’s clinical score >50 %, whatever the KCCQ score value. When the KCCQ score was ≤50 %, there was a low increase in risk as the Pocock’s clinical score increased (OR 2.0 [0.6; 6.6]). However, when the KCCQ score was between 50 and 75 or ≥75 %, there was a high increase in risk as the Pocock’s clinical score increased (OR 6.9 [1.2; 38.9] and OR 7.4 [0.8; 69.7], respectively). // Conclusions : Patients with a high Pocock’s clinical score are at a high risk of death or hospitalization. For patients with a low Pocock’s clinical score, the KCCQ score can identify those at risk of these events.

Xenotransplantation of human cells in animal models is an essential tool for evaluation of safety and efficacy of cell-based products for therapeutic use. Sensitive and reproducible methods are needed to detect and quantify human cells engrafted into the host tissue either in the targeted organ or in undesired locations. We developed a robust quantitative polymerase chain reaction (qPCR) assay based on amplification of human AluYb8 repeats, to assess the number of human cells present in rat or mouse tissues after transplantation. Standard curves of mixed human/rodent DNA and mixed human/rodent cells have been performed to determine the limit of detection and linear range of the assay. Standard curves from DNA mixing differed significantly from standard curves from cell mixing. We show here that the AluYb8 qPCR assay is highly reproducible and is able to quantify human cells in a rodent cell matrix over a large linear range that extends from 50 to 0.01% human cells. Short-term in vivo studies showed that human cells could be quantified in mouse liver up to 7 days after intra-splenic transplantation and in rat liver four hours after intra-hepatic transplantation.

STUDY QUESTION: What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh? SUMMARY ANSWER: Ovarian cells from fresh medullary tissue, which can be isolated in larger numbers, show higher viability and are able to improve graft vascularization. WHAT IS KNOWN ALREADY: In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells. STUDY DESIGN, SIZE, DURATION: Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cryopreservation decreased ovarian cell yield (-2804 cells/mg, P = 0.015) and viability (-9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. METHODS: In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). RESULTS: All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). CONCLUSIONS: This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while provid

PURPOSE: The aim of this study was to determine the best combination in terms of cryopreservation techniques and vascular bed preparation before grafting in order to obtain functional ovarian tissue after transplantation. METHODS: Five cynomolgus monkeys were used. Strips from 10 ovaries were cryopreserved, 5 by vitrification (V), and 5 by slow-freezing (SF). Pieces of fresh ovarian tissue were used for controls. After 1 month, the strips were autografted to two different vascular beds, healed (HB) or freshly decorticated (FDB), constituting four study groups: SF-HB, SF-FDB, V-HB, and V-FDB. These were compared to fresh tissue. After 6 months, the ovaries were removed and several parameters analyzed: follicle quality, stage, density, proliferation, apoptosis, functionality, vascularization, and fibrosis. Mixed effect linear regression models were built to assess the impact of cryopreservation and vascular bed preparation on ovarian tissue viability and functionality. p values were adjusted for multiple testing using the Benjamini-Hochberg method, and q values < 0.20 were considered significant in order to achieve a 20% false discovery rate. RESULTS: Compared to fresh tissue, no difference was observed in the percentage of morphologically normal follicles, while a significant increase was noted in the follicle proliferation rate (41%, q = 0.19), percentage of antral follicles (12%, q = 0.14), and number of vessels per area (3.3 times, q = 0.07) in the V-FDB group. CONCLUSIONS: Vitrification associated with FDB vascular bed preparation is the best combination to obtain functional autografted ovarian tissue. Further studies are nevertheless required, with confirmed pregnancies and live births before introducing the procedure into clinical practice.

AIMS: Slide digitalization has brought pathology to a new era, including powerful image analysis possibilities. However, while being a powerful prognostic tool, immunostaining automated analysis on digital images is still not implemented worldwide in routine clinical practice. METHODS AND RESULTS: Digitalized biopsy sections from two independent cohorts of patients, immunostained for membrane or nuclear markers, were quantified with two automated methods. The first was based on stained cell counting through tissue segmentation, while the second relied upon stained area proportion within tissue sections. Different steps of image preparation, such as automated tissue detection, folds exclusion and scanning magnification, were also assessed and validated. Quantification of either stained cells or the stained area was found to be correlated highly for all tested markers. Both methods were also correlated with visual scoring performed by a pathologist. For an equivalent reliability, quantification of the stained area is, however, faster and easier to fine-tune and is therefore more compatible with time constraints for prognosis. CONCLUSIONS: This work provides an incentive for the implementation of automated immunostaining analysis with a stained area method in routine laboratory practice.