Multiomics approach to profiling Sertoli cell maturation during development of the spermatogonial stem cell niche.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis, a complex process supported by a specialized microenvironment, called the SSC niche. Postnatal development of SSCs is characterized by distinct metabolic transitions from prepubertal to adult stages. An understanding of the niche factors that regulate these maturational events is critical for the clinical application of SSCs in fertility preservation. To investigate the niche maturation events that take place during SSC maturation, we combined different '-omics' technologies. Serial single cell RNA sequencing analysis revealed changes in the transcriptomes indicative of niche maturation that was initiated at 11 years of age in humans and at 8 weeks of age in pigs, as evident by Monocle analysis of Sertoli cells and peritubular myoid cell (PMC) development in humans and Sertoli cell analysis in pigs. Morphological niche maturation was associated with lipid droplet accumulation, a characteristic that was conserved between species. Lipidomic profiling revealed an increase in triglycerides and a decrease in sphingolipids with Sertoli cell maturation in the pig model. Quantitative (phospho-) proteomics analysis detected the activation of distinct pathways with porcine Sertoli cell maturation. We show here that the main aspects of niche maturation coincide with the morphological maturation of SSCs, which is followed by their metabolic maturation. The main aspects are also conserved between the species and can be predicted by changes in the niche lipidome. Overall, this knowledge is pivotal to establishing cell/tissue-based biomarkers that could gauge stem cell maturation to facilitate laboratory techniques that allow for SSC transplantation for restoration of fertility.

Metabolic transitions define spermatogonial stem cell maturation.

STUDY QUESTION: Do spermatogonia, including spermatogonial stem cells (SSCs), undergo metabolic changes during prepubertal development? SUMMARY ANSWER: Here, we show that the metabolic phenotype of prepubertal human spermatogonia is distinct from that of adult spermatogonia and that SSC development is characterized by distinct metabolic transitions from oxidative phosphorylation (OXPHOS) to anaerobic metabolism. WHAT IS KNOWN ALREADY: Maintenance of both mouse and human adult SSCs relies on glycolysis, while embryonic SSC precursors, primordial germ cells (PGCs), exhibit an elevated dependence on OXPHOS. Neonatal porcine SSC precursors reportedly initiate a transition to an adult SSC metabolic phenotype at 2 months of development. However, when and if such a metabolic transition occurs in humans is ambiguous. STUDY DESIGN, SIZE, DURATION: To address our research questions: (i) we performed a meta-analysis of publicly available and newly generated (current study) single-cell RNA sequencing (scRNA-Seq) datasets in order to establish a roadmap of SSC metabolic development from embryonic stages (embryonic week 6) to adulthood in humans (25 years of age) with a total of ten groups; (ii) in parallel, we analyzed single-cell RNA sequencing datasets of isolated pup (n = 3) and adult (n = 2) murine spermatogonia to determine whether a similar metabolic switch occurs; and (iii) we characterized the mechanisms that regulate these metabolic transitions during SSC maturation by conducting quantitative proteomic analysis using two different ages of prepubertal pig spermatogonia as a model, each with four independently collected cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single testicular cells collected from 1-year, 2-year and 7-year-old human males and sorted spermatogonia isolated from 6- to 8-day (n = 3) and 4-month (n = 2) old mice were subjected to scRNA-Seq. The human sequences were individually processed and then merged with the publicly available datasets for a meta-analysis using Seurat V4 package. We then performed a pairwise differential gene expression analysis between groups of age, followed by pathways enrichment analysis using gene set enrichment analysis (cutoff of false discovery rate < 0.05). The sequences from mice were subjected to a similar workflow as described for humans. Early (1-week-old) and late (8-week-old) prepubertal pig spermatogonia were analyzed to reveal underlying cellular mechanisms of the metabolic shift using immunohistochemistry, western blot, qRT-PCR, quantitative proteomics, and culture experiments. MAIN RESULTS AND THE ROLE OF CHANCE: Human PGCs and prepubertal human spermatogonia show an enrichment of OXPHOS-associated genes, which is downregulated at the onset of puberty (P < 0.0001). Furthermore, we demonstrate that similar metabolic changes between pup and adult spermatogonia are detectable in the mouse (P < 0.0001). In humans, the metabolic transition at puberty is also preceded by a drastic change in SSC shape at 11 years of age (P < 0.0001). Using a pig model, we reveal that this metabolic shift could be regulated by an insulin growth factor-1 dependent signaling pathway via mammalian target of rapamycin and proteasome inhibition. LARGE SCALE DATA: New single-cell RNA sequencing datasets obtained from this study are freely available through NCBI GEO with accession number GSE196819. LIMITATIONS, REASONS FOR CAUTION: Human prepubertal tissue samples are scarce, which led to the investigation of a low number of samples per age. Gene enrichment analysis gives only an indication about the functional state of the cells. Due to limited numbers of prepubertal human spermatogonia, porcine spermatogonia were used for further proteomic and in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: We show that prepubertal human spermatogonia exhibit high OXHPOS and switch to an adult-like metabolism only after 11 years of age. Prepubescent cancer survivors often suffer from infertility in adulthood. SSC transplantation could provide a powerful tool for the treatment of infertility; however, it requires high cell numbers. This work provides key insight into the dynamic metabolic requirements of human SSCs across development that would be critical in establishing ex vivo systems to support expansion and sustained function of SSCs toward clinical use. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the NIH/NICHD R01 HD091068 and NIH/ORIP R01 OD016575 to I.D. K.E.O. was supported by R01 HD100197. S.K.M. was supported by T32 HD087194 and F31 HD101323. The authors declare no conflict of interest.

Long-Term Aspirin Use and Self-Reported Walking Speed in Older Men: The Physicians' Health Study.

BACKGROUND: Mobility limitation is a component of frailty that shares a bidirectional relationship with cardiovascular disease (CVD). Data are limited on the role of established CVD prevention therapies, such as aspirin, for prevention of frailty and mobility limitation. OBJECTIVES: Examine the association between long-term aspirin use and walking speed. DESIGN, SETTING, PARTICIPANTS: Prospective cohort of 14,315 men who participated in the Physicians' Health Study I, a completed randomized controlled trial of aspirin (1982-1988), with extended post-trial follow-up. MEASUREMENTS: Annual questionnaires collected data on aspirin use, lifestyle and other factors. Average annual aspirin use was categorized for each participant: ≤60 days/year and >60 days/year. Mobility was defined according to self-reported walking pace, categorized as: don't walk regularly (reference), easy/casual <2mph, normal ≥2-2.9mph, or brisk/very brisk ≥3mph. Propensity scoring balanced covariates between aspirin categories. Multinomial logistic regression models estimated odds of being in each self-reported walking category. RESULTS: Mean age was 70±8 years; mean aspirin use 11 years. There were 2,056 (14.3%) participants who reported aspirin use ≤60 days/year. Aspirin use >60 days/year was associated with drinking alcohol, smoking, hypertension, heart disease and stroke, while ≤60 days/year was associated with anticoagulation use and bleeding history. In all, 13% reported not walking regularly, 12% walked <2 mph, 44% walked ≥2-2.9 mph, and 31% walked ≥3 mph. After propensity score adjustment, regular aspirin use was associated with a faster walking speed. Odds ratios (95% confidence intervals) were 1.16 (0.97 to 1.39), 1.24 (1.08 to 1.43), and 1.40 (1.21 to 1.63) for <2 mph, ≥2-2.9 mph and ≥3 mph, respectively, compared to not walking regularly (p-trend<0.001). CONCLUSIONS: In this cohort of older men, long-term aspirin use is associated with a greater probability of faster walking speed later in life.

MMP8 increases tongue carcinoma cell-cell adhesion and diminishes migration via cleavage of anti-adhesive FXYD5.

Matrix metalloproteinases (MMPs) modify bioactive factors via selective processing or degradation resulting in tumour-promoting or tumour-suppressive effects, such as those by MMP8 in various cancers. We mapped the substrates of MMP8 to elucidate its previously shown tumour-protective role in oral tongue squamous cell carcinoma (OTSCC). MMP8 overexpressing (+) HSC-3 cells, previously demonstrated to have reduced migration and invasion, showed enhanced cell-cell adhesion. By analysing the secretomes of MMP8 + and control cells with terminal amine isotopic labelling of substrates (TAILS) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS), we identified 36 potential substrates of MMP8, including FXYD domain-containing ion transport regulator 5 (FXYD5). An anti-adhesive glycoprotein FXYD5 has been previously shown to predict poor survival in OTSCC. Cleavage of FXYD5 by MMP8 was confirmed using recombinant proteins. Furthermore, we detected a loss of FXYD5 levels on cell membrane of MMP8 + cells, which was rescued by inhibition of the proteolytic activity of MMP8. Silencing (si) FXYD5 increased the cell-cell adhesion of control but not that of MMP8 + cells. siFXYD5 diminished the viability and motility of HSC-3 cells independent of MMP8 and similar effects were seen in another tongue cancer cell line, SCC-25. FXYD5 is a novel substrate of MMP8 and reducing FXYD5 levels either with siRNA or cleavage by MMP8 increases cell adhesion leading to reduced motility. FXYD5 being a known prognostic factor in OTSCC, our findings strengthen its potential as a therapeutic target.

Dietary patterns of French adults: associations with demographic, socio-economic and behavioural factors.

BACKGROUND: Although the French eating model may differ from those of other countries, no studies to date have investigated dietary patterns in a wide age range of adults and at the national level. We aimed to identify dietary patterns (DP) of French adults and assess their associations with demographic, socio-economic and behavioural factors. METHODS: The present study included 2624 adults (1087 men, 1537 women) aged 18-79 years from the cross-sectional national French INCA2 dietary survey. Dietary data were collected using a 7-day estimated food record. Clusters of DP were derived using principal component analysis and clustering, conjointly. Age-adjusted logistic regression analyses were used to investigate the association between DP and correlates. RESULTS: Five DP were identified, namely 'traditional', 'prudent', 'diversified', 'processed' and 'sandwiches'. Men were more likely to follow a traditional diet and women the 'prudent' pattern. Members of the 'processed' and 'sandwiches' patterns were younger compared to non-members. Healthier dietary patterns were overall positively associated with a higher socio-economic position, healthier behaviours (in terms of sedentary behaviours and smoking status) and lower body mass index. Under-reporting of energy intake, restrictive diet to lose weight and dietary supplement consumption were also related to specific DP, although differentially in men and women. Associations with contextual factors (i.e. household composition, agglomeration size and region) were also observed. CONCLUSIONS: The identification of adults' dietary patterns and associated behaviours (all modifiable) is important for the conceptualisation of multi-behavioural programs. The additional information on social and environmental correlates is also essential for targeting the most vulnerable population groups in the context of such public health interventions.

Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease. Various disease-related and patient-related factors have been shown to influence the course of the disease. The aim of this study was to identify novel biomarkers of significant clinical relevance. Pretreatment CD19-separated lymphocytes (n=237; discovery set) and peripheral blood mononuclear cells (n=92; validation set) from the REACH trial, a randomized phase III trial in relapsed CLL comparing rituximab plus fludarabine plus cyclophosphamide with fludarabine plus cyclophosphamide alone, underwent gene expression profiling. By using Cox regression survival analysis on the discovery set, we identified inositol polyphosphate-5-phosphatase F (INPP5F) as a prognostic factor for progression-free survival (P<0.001; hazard ratio (HR), 1.63; 95% confidence interval (CI), 1.35-1.98) and overall survival (P<0.001; HR, 1.47; 95% CI, 1.18-1.84), regardless of adjusting for known prognostic factors. These findings were confirmed on the validation set, suggesting that INPP5F may serve as a novel, easy-to-assess future prognostic biomarker for fludarabine-based therapy in CLL.

Management of the extravasation of anti-neoplastic agents.

BACKGROUND: Extravasation is a potentially severe complication that can occur during the administration of chemotherapy. The scarcity of evidence available makes it difficult to develop an optimal management scheme. The purpose of this guideline is to review the relevant scientific literature on the prevention, management, and treatment of extravasation occurring during the administration of chemotherapy to cancer patients. METHOD: A scientific literature review was conducted using the PubMed search tool. The period covered was from database inception to April 2014, inclusively. Since the literature on extravasation treatment is often empirical, anecdotal, and controversial, the review also identified clinical practice guidelines and expert consensuses published by relevant international organizations and cancer agencies. RESULTS: Identification of potential risk factors and preventive measures can reduce the risk of extravasation. Recognition and management of symptoms are crucial in patients with this complication. Provision of adequate instruction to personnel responsible for administering chemotherapy and to patients on recognizing symptoms, preventing, and managing extravasation is essential. Extravasation can be treated with dry warm or cold compresses and various antidotes such as dimethyl sulfoxide, dexrazoxane, hyaluronidase, or sodium thiosulfate, depending on the agent that has caused extravasation. Patient monitoring to assess the progression or regression of symptoms and to thus take the appropriate measures is necessary. CONCLUSION: Several strategies must be established to ensure that extravasation is recognized and properly managed. Given the evidence available at this time, the Comité de l'évolution des pratiques en oncologie (CEPO) has made recommendations for clinical practice in Quebec.

Dual PI3K/mTOR inhibition shows antileukemic activity in MLL-rearranged acute myeloid leukemia.

In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Early assessment of minimal residual disease in AML by flow cytometry during aplasia identifies patients at increased risk of relapse.

In acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) by flow cytometry (flow MRD) after induction and consolidation therapy has been shown to provide independent prognostic information. However, data on the value of earlier flow MRD assessment are lacking. Therefore, the value of flow MRD detection was determined during aplasia in 178 patients achieving complete remission after treatment according to AMLCG (AML Cooperative Group) induction protocols. Flow MRD positivity during aplasia predicted poor outcome (5-year relapse-free survival (RFS) 16% vs 43%, P<0.001) independently from age and cytogenetic risk group (hazard ratio for MRD positivity 1.71; P=0.009). Importantly, the prognosis of patients without detectable MRD was neither impacted by morphological blast count during aplasia nor by MRD status postinduction. Early flow MRD was also evaluated in the context of existing risk factors. Flow MRD was prognostic within the intermediate cytogenetic risk group (5-year RFS 15% vs 37%, P=0.016) as well as for patients with normal karyotype and NPM1 mutations (5-year RFS 13% vs 49%, P=0.02) or FLT3-ITD (3-year RFS rates 9% vs 44%, P=0.016). Early flow MRD assessment can improve current risk stratification approaches by prediction of RFS in AML and might facilitate adaptation of postremission therapy for patients at high risk of relapse.

Associations between usual school lunch attendance and eating habits and sedentary behaviour in French children and adolescents.

BACKGROUND/OBJECTIVE: Our objective was to investigate whether school lunch attendance was associated with overall eating habits and sedentary behaviour in a French sample of children and adolescents. SUBJECTS/METHODS: Data for the study were taken from the second French cross-sectional dietary survey (INCA2-2006-07). In total, 1413 school children aged 3-17 years old were classified according to their school type and their usual school lunch attendance. Eating habits included meal regularity, dietary diversity, purchase in vending machine, snacking habits and frequency of eating in fast-foods. Two composite indices of eating habits were derived from multiple correspondence analyses. Sedentary behaviour was assessed by the average daily screen times for TV and computer. The association between school lunch attendance and each variable was tested. Multivariate association between school lunch attendance and the composite indices of eating habits and sedentary behaviours was studied. RESULTS: In all, 69.0% (CI(95%): 64.2-73.9) of secondary school children and 63.0% (CI(95%): 58.5-67.5) of pre- and elementary school children usually attended school lunch at least once a week. Pre- and elementary school children attending school lunches showed a higher dietary diversity score (P=0.02) and ate morning snacks more frequently (P=0.02). In secondary school children, attending school canteen was related to a lower rate of skipping breakfast (P=0.04) and main meals (P=0.01). In all school children, school lunch attendance was simultaneously associated with healthier overall eating habits and less sedentary behaviour. CONCLUSION: In France, children attending school canteens seem to have healthier eating habits and display less sedentary behaviour, independently of their socio-economic and demographic background.

Identification of recurring tumor-specific somatic mutations in acute myeloid leukemia by transcriptome sequencing.

Genetic lesions are crucial for cancer initiation. Recently, whole genome sequencing, using next generation technology, was used as a systematic approach to identify mutations in genomes of various types of tumors including melanoma, lung and breast cancer, as well as acute myeloid leukemia (AML). Here, we identify tumor-specific somatic mutations by sequencing transcriptionally active genes. Mutations were detected by comparing the transcriptome sequence of an AML sample with the corresponding remission sample. Using this approach, we found five non-synonymous mutations specific to the tumor sample. They include a nonsense mutation affecting the RUNX1 gene, which is a known mutational target in AML, and a missense mutation in the putative tumor suppressor gene TLE4, which encodes a RUNX1 interacting protein. Another missense mutation was identified in SHKBP1, which acts downstream of FLT3, a receptor tyrosine kinase mutated in about 30% of AML cases. The frequency of mutations in TLE4 and SHKBP1 in 95 cytogenetically normal AML patients was 2%. Our study demonstrates that whole transcriptome sequencing leads to the rapid detection of recurring point mutations in the coding regions of genes relevant to malignant transformation.

One year variability of peak heights, heterozygous balance and inter-locus balance for the DNA positive control of AmpFℓSTR© Identifiler© STR kit.

Multivariate analyses of 205 positive control experiments in an AmpFℓSTR© Identifiler© STR kit were used to analyze the factors affecting peak heights at 16 loci. Peak heights were found to be highly correlated between loci and there was evidence for a difference in sensitivity of the two genetic analyzers in the blue channel. Heterozygous balance response at 10 loci was found to behave as a random variable following a beta-distribution with typical median values of 90%, without locus or genetic analyzer effect. Inter-locus balance at 16 loci was influenced by the blue channel effect and a temporal switch of unexplained origin. The implications of these results for the choice of minimum threshold values in quality control are discussed.

Thermal removal of nitrogen species from wood waste containing urea formaldehyde and melamine formaldehyde resins.

The removal of nitrogen from wood board waste through a low temperature pyrolysis (523-573 K) is investigated with two analytical methods. The kinetic study of the thermal behaviour of wood board and of its components (wood, UF and MF resins) shows the feasibility of removing thermally nitrogen from wood board waste. Indeed, the range of temperatures associated with the degradation of wood is different from the one obtained for the degradation of UF and MF resin. Isothermal conditions enable the determination of a kinetic model for degradation of wood board and of its components and demonstrate that the thermal behaviour of wood board is not the reflection of the sum of its components' behaviour. FTIR analysis of gas products confirms the feasibility removing nitrogen thermally and enables the evaluation of the optimum treatment conditions (temperature/duration). Elementary analysis of the treated samples and study of their low heating value (LHV) enable to quantify the efficiency of the thermal treatment in terms of nitrogen removal and of energy recovery. Results show that around 70% of the initial nitrogen can be removed from the waste, and that the temperature of treatment (between 523 K and 573 K) does not influence the efficiency in terms of nitrogen removal. Nevertheless, the ratio Residual energy/Initial energy (between 76% and 90%) is improved with the lowest temperature of treatment.

Eigenanalysis of selection ratios from animal radio-tracking data.

The development of methods to analyze habitat selection when resources are defined by several categories (e.g., vegetation types) is a topical issue in radio-tracking studies. The White and Garrott statistic, an extension of the widely used test of Neu et al., can be used to determine whether habitat selection is significant. As well, Manly's selection ratio, a particularly useful measure of resource selectivity by resource users, allows detection of the most strongly selected habitat types. However, when both the number of animals and types of habitat are large, the biologist often has to deal with an excessively large number of measures. In this paper we present a new method, the eigenanalysis of selection ratios, that generalizes these two common methods within the framework of eigenanalyses. This method undertakes an additive linear partitioning of the White and Garrott statistic, so that the difference between habitat use and availability is maximized on the first factorial axes. The eigenanalysis of selection ratios is therefore optimal in habitat selection studies. Although we primarily consider the case where the habitat availability is the same for all animals (design II), we also extend this analysis to the case where the habitat availability varies from one animal to another (design III). An application of this method is provided using radio-tracking data collected on 17 squirrels in five habitat types. The results indicate variability in habitat selection, with two groups of animals displaying two patterns of preference. This difference between the two groups is explained by the patch structure of the study area. Because this method is mainly exploratory, and therefore does not rely on any distributional assumption, we recommend its use in studies of habitat selection.

Report on the Belgian consensus meeting on colorectal cancer screening.

Screening and prevention of colorectal cancer must be a public health priority. It is the most frequent malignancy in Europe, the second leading cause of cancer death, including Belgium where more than 6000 new cases occur per year. Various screening modalities, from non invasive to invasive are available and currently in use and they are all cost-effective in comparison with no screening. The decision as to which screening test to use should be made by the patient and clinician. Consensus documents prepared by the Belgian scientific community appear in this issue of Acta Gastroenterologica Belgica, summarizing the scientific evidence in favour as well as the limitations of fecal occult blood tests, flexible sigmoidoscopy, videocolonoscopy and virtual colonoscopy.

Association of hepatitis B virus infection with other sexually transmitted infections in homosexual men. Omega Study Group.

OBJECTIVES: This study determined the prevalence and factors associated with hepatitis B virus (HBV) infection among men who have sex with men. METHODS: At the baseline visit of an HIV study among men who have sex with men, we asked about HBV vaccination status and tested for HBV markers. RESULTS: Of 625 subjects, 48% had received at least 1 dose of HBV vaccine. Of 328 unvaccinated men, 41% had 1 or more HBV markers. HBV prevalence increased markedly with age and was associated with many sexual and drug-related behaviors. In a multivariate model, 7 variables were independently associated with HBV infection: ulcerative sexually transmitted diseases (odds ratio [OR] = 10.1; 95% confidence interval [CI] = 2.6, 54); injection drug use (OR = 5.2; 95% CI = 1.2, 26); gonorrhea or chlamydia (OR = 4.0; 95% CI = 1.9, 8.9); sexual partner with HIV/AIDS (OR = 3.6; 95% CI = 1.8, 7.1); 50 or more casual partners (OR = 3.4; 95% CI = 1.6, 7.1); received money for sex (OR = 3.0; 95% CI = 1.2, 7.8); and 20 or more regular partners (OR = 2.5; 95% CI = 1.1, 6.1). CONCLUSIONS: In Montreal, men who have sex with men are at risk for HBV infection, but a substantial proportion remain unvaccinated; new strategies are required to improve coverage. Men who have sex with men and who have a sexually transmitted infection, especially a genito-ulcerative infection, appear to be at particularly high risk for HBV infection.

High-dose methylprednisolone reduces cytokine-induced adhesion molecules on human brain endothelium.

OBJECTIVE: We investigated the in vitro effects of low- and high-dose methylprednisolone (MP) on the cytokine-induced expression of HLA-DR, ICAM-1 and VCAM-1 on human brain microvessel endothelial cells (HBMECs). METHODS: Brain endothelium was obtained from microvessels included in the apparently normal white matter of surgical specimens of nine patients. Cells were stained with monoclonal antibodies anti-HLA-DR, anti-ICAM-1 and anti-VCAM-1 and analysed by flow cytometry as fluorescence histograms. The mean fluorescence intensity (MFI) of HBMECs treated with different stimuli was calculated. RESULTS: gamma-IFN-induced HLA-DR was down-regulated in a dose-dependent manner by MP. High-dose MP reduced the TNF-alpha-induced ICAM-1 and VCAM-1 expression. CONCLUSIONS: The down-regulation of adhesion molecules on cerebral endothelial cells could decrease mononuclear cell transmigration through the blood brain barrier and consequently the perivascular infiltrates. The results add support to the rationale for high-dose MP treatment in multiple sclerosis relapses.