Characterizing the pathotype of neonatal meningitis causing Escherichia coli (NMEC).

BACKGROUND: Neonatal meningitis-causing Escherichia coli (NMEC) is the predominant Gram-negative bacterial pathogen associated with meningitis in newborn infants. High levels of heterogeneity and diversity have been observed in the repertoire of virulence traits and other characteristics among strains of NMEC making it difficult to define the NMEC pathotype. The objective of the present study was to identify genotypic and phenotypic characteristics of NMEC that can be used to distinguish them from commensal E. coli. METHODS: A total of 53 isolates of NMEC obtained from neonates with meningitis and 48 isolates of fecal E. coli obtained from healthy individuals (HFEC) were comparatively evaluated using five phenotypic (serotyping, serum bactericidal assay, biofilm assay, antimicorbial susceptibility testing, and in vitro cell invasion assay) and three genotypic (phylogrouping, virulence genotyping, and pulsed-field gel electrophoresis) methods. RESULTS: A majority (67.92%) of NMEC belonged to B2 phylogenetic group whereas 59% of HFEC belonged to groups A and D. Serotyping revealed that the most common O and H types present in NMEC tested were O1 (15%), O8 (11.3%), O18 (13.2%), and H7 (25.3%). In contrast, none of the HFEC tested belonged to O1 or O18 serogroups. The most common serogroup identified in HFEC was O8 (6.25%). The virulence genotyping reflected that more than 70% of NMEC carried kpsII, K1, neuC, iucC, sitA, and vat genes with only less than 27% of HFEC possessing these genes. All NMEC and 79% of HFEC tested were able to invade human cerebral microvascular endothelial cells. No statistically significant difference was observed in the serum resistance phenotype between NMEC and HFEC. The NMEC strains demonstrated a greater ability to form biofilms in Luria Bertani broth medium than did HFEC (79.2% vs 39.9%). CONCLUSION: The results of our study demonstrated that virulence genotyping and phylogrouping may assist in defining the potential NMEC pathotype.

BBB on chip: microfluidic platform to mechanically and biochemically modulate blood-brain barrier function.

The blood-brain barrier (BBB) is a unique feature of the human body, preserving brain homeostasis and preventing toxic substances to enter the brain. However, in various neurodegenerative diseases, the function of the BBB is disturbed. Mechanisms of the breakdown of the BBB are incompletely understood and therefore a realistic model of the BBB is essential. We present here the smallest model of the BBB yet, using a microfluidic chip, and the immortalized human brain endothelial cell line hCMEC/D3. Barrier function is modulated both mechanically, by exposure to fluid shear stress, and biochemically, by stimulation with tumor necrosis factor alpha (TNF-α), in one single device. The device has integrated electrodes to analyze barrier tightness by measuring the transendothelial electrical resistance (TEER). We demonstrate that hCMEC/D3 cells could be cultured in the microfluidic device up to 7 days, and that these cultures showed comparable TEER values with the well-established Transwell assay, with an average (± SEM) of 36.9 Ω.cm(2) (± 0.9 Ω.cm(2)) and 28.2 Ω.cm(2) (± 1.3 Ω.cm(2)) respectively. Moreover, hCMEC/D3 cells on chip expressed the tight junction protein Zonula Occludens-1 (ZO-1) at day 4. Furthermore, shear stress positively influenced barrier tightness and increased TEER values with a factor 3, up to 120 Ω.cm(2). Subsequent addition of TNF-α decreased the TEER with a factor of 10, down to 12 Ω.cm(2). This realistic microfluidic platform of the BBB is very well suited to study barrier function in detail and evaluate drug passage to finally gain more insight into the treatment of neurodegenerative diseases.

Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.

The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1ß or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs).

Differential activation of mitochondrial apoptotic pathways by vasculotropic amyloid-beta variants in cells composing the cerebral vessel walls.

Cerebral amyloid angiopathy (CAA) is an age-associated condition and a common finding in Alzheimer's disease in which amyloid-beta (Abeta) vascular deposits are featured in >80% of the cases. Familial Abeta variants bearing substitutions at positions 21-23 are primarily associated with CAA, although they manifest with strikingly different clinical phenotypes: cerebral hemorrhage or dementia. The recently reported Piedmont L34V Abeta mutant, located outside the hot spot 21-23, shows a similar hemorrhagic phenotype, albeit less aggressive than the widely studied Dutch E22Q variant. We monitored the apoptotic events occurring after stimulation of human brain microvascular endothelial and smooth muscle cells with nonfibrillar structures of both variants and wild-type Abeta40. Induction of analogous caspase-mediated mitochondrial pathways was elicited by all peptides, although within different time frames and intensity. Activated pathways were susceptible to pharmacological modulation either through direct inhibition of mitochondrial cytochrome c release or by the action of pan- and pathway-specific caspase inhibitors, giving a clear indication of the independent or synergistic engagement of both extrinsic and intrinsic mechanisms. Structural analyses of the Abeta peptides showed that apoptosis preceded fibril formation, correlating with the presence of oligomers and/or protofibrils. The data support the notion that rare genetic mutations constitute unique paradigms to understand the molecular pathogenesis of CAA.

Calcein assay: a high-throughput method to assess P-gp inhibition.

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.

Cyclical antiidiotypic response to anti-hormone antibodies due to neutralization by autologous anti-antiidiotype antibodies that bind hormone.

Antiidiotype antibodies were raised against anti-catecholamine ligand antibodies. The antiidiotype response was shown to be cyclical and to correspond to the production of antibodies that could bind to catecholamine beta-adrenergic receptors and stimulate adenylate cyclase. Disappearance of these antibodies from the serum could be correlated with the appearance of a catecholamine ligand-binding activity corresponding to the synthesis of autologous anti-antiidiotype antibodies directed against the induced antiidiotypic molecules. Comparison of the injected versus the induced anti-ligand antibodies reveals striking differences in affinities but similarities in the ability to bind to the antiidiotype antibodies and to the ligand-containing affinity gel. The results support the existence of a functional network of idiotype antiidiotype interactions involving external as well as internal antigens, antibodies, and possibly other types of molecules involved in recognition phenomena, such as hormone receptors.

[Blood-brain barrier part III: therapeutic approaches to cross the blood-brain barrier and target the brain]. / Barrière hématoencéphalique Partie III : approche thérapeutique pour franchir la barrière hématoencéphalique.

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches.

Molecular cytogenetic characterization of the human cerebral microvessel endothelial cell line hCMEC/D3.

The immortalized human cerebral microvessel endothelial cell line hCMEC/D3 has been repeatedly used as a model of human blood-brain barrier (BBB). hCMEC/D3 cells between passage 25 and 35 are most often applied in research, remained phenotypically nontransformed, and cells maintained many characteristics of human brain endothelial cells. Also hCMEC/D3 was thought to have conserved a normal diploid karyotype over all these passages. Here we characterized the cell line using high-resolution multicolor fluorescence in situ hybridization (FISH) approaches and revealed a complex karyotype in the 30th passage. Clonal cryptic unbalanced structural rearrangements and numerical aberrations were discovered and described as follows: 45 approximately 48,XX, -X,del(5)(q11)[2],del(9)(q11)[3],+9[3],del(11)(q13 approximately 14)[2], der(14)t(14;21)(q32.33;q22.3)[28],der(15)t(9;15)(p11;p11)[13], dup(15)(p11q11)[5],der(21)t(17;21)(p12;q22)[9],-22[6][cp28]. In summary, a complex karyotype with clonal unbalanced chromosomal rearrangements is present in hCMEC/D3. Thus, we solicit to include molecular cytogenetics in the testing of all cell lines prior to application of their use in complex studies.

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis.

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.

[Biology of the blood-brain barrier: Part I]. / Biologie de la barrière hématoencéphalique : Partie I.

The blood-brain barrier provides the central nervous system with a unique protection against the toxic effects of many xenobiotics. This protection results from the unique anatomic and biological structure of the endothelium of blood vessels in the brain. The main features of the blood-brain barrier are the presence of tight intercellular junctions which strictly limit the diffusion of blood-borne solutes and cells into the brain and the polarized expression of transporters which specifically control the cerebral availability of nutrients, drugs or xenobiotics. Recent findings in molecular and cellular biology improved our knowledge of blood-brain barrier permeability and its regulation. The importance of these findings has been recently highlighted by the description of dysfunctions of the blood-brain barrier which could have an impact on the pathophysiology of several neurological diseases. This review focuses on recent advances in our understanding of blood-brain barrier biology and physiology, presenting the structural organization of the blood-brain barrier and the functional regulation of solute permeability and cellular transendothelial migration.

[Implication of the blood-brain barrier in neurological diseases: part II]. / Implication de la barrière hématoencéphalique dans la physiopathologie des maladies neurologiques : Partie II.

The main characteristic of the blood-brain-barrier (BBB) is its extremely low permeability, due to tight intercellular endothelial junctions and a variety of transporters, which provides the brain with a unique protection against the potential toxicity of several xenobiotics, but also constitutes a major limitation to drug delivery to the central nervous system. Several dysfunctions of the BBB have been recently implicated in the pathophysiology of neurological diseases: inflammatory, vascular, tumoral, infectious and neurodegenerative diseases. Based on a better knowledge of the BBB biology, new therapeutic strategies are emerging, which by-pass the BBB or take advantage of the selective expression of membrane proteins by brain endothelial cells or circulating leucocytes to target new drugs, such as the anti-VLA4 antibody recently approved for multiple sclerosis treatment. This review will focus on the recently described BBB dysfunctions presumably involved in various neurological diseases.

Circulating endothelial progenitor cells in systemic sclerosis: association with disease severity.

BACKGROUND: Heterogeneous data have been reported regarding the detection and number of circulating endothelial progenitor cells (EPCs) in systemic sclerosis (SSc). OBJECTIVE: We investigated the number of circulating EPCs using recent recommendations and we quantified their late outgrowth in patients with SSc and healthy controls. PATIENTS AND METHODS: EPCs, defined as Lin-/7AAD-/CD34+/CD133+/VEGFR-2+ cells, were quantified in 50 patients with SSc (mean age: 55 (16) years, disease duration: 9 (9) years) and 26 controls (mean age: 53 (19) years) by cell sorting/flow cytometry and by counting late outgrowth colony-forming units (CFU). RESULTS: Patients with SSc displayed higher circulating EPC counts than controls (median 86 (5-282) vs 49 (5-275)) EPCs for 1 million Lin- mononuclear cells; p = 0.01). Lower EPC counts were associated with the higher Medsger's severity score (p = 0.01) and with the presence of past and/or current digital ulcers (p = 0.026). There was no difference for the number of late outgrowth EPC-CFUs between patients with SSc and controls in cell culture evaluation. The formation of colonies was associated with higher levels of circulating EPCs (p = 0.02) and the number of colonies correlated with levels of EPCs (R = 0.73, p = 0.0004), validating our combination of fluorescence-activated cell sorter surface markers. CONCLUSIONS: We quantified circulating EPCs with an accurate combination of markers herein validated. Our data demonstrate increased circulating EPC levels in SSc, supporting their mobilisation from bone marrow. Furthermore, the subset of patients with digital vascular lesions and high severity score displayed low EPC counts, suggesting increased homing at this stage. The predictive value of this biomarker now warrants further evaluation.

Structural organization and expression of human MTUS1, a candidate 8p22 tumor suppressor gene encoding a family of angiotensin II AT2 receptor-interacting proteins, ATIP.

The Mitochondrial Tumor suppressor 1 (MTUS1) gene is a newly identified candidate tumor suppressor gene at chromosomal position 8p22. We report here that MTUS1 encodes a family of proteins whose leader member (ATIP1) was previously isolated in our laboratory as a novel interacting partner of the angiotensin II AT2 receptor involved in growth inhibition (Nouet, JBC 279: 28989-97, 2004). The MTUS1 gene contains 17 coding exons distributed over 112 kb of genomic DNA. Alternative exon usage generates three major transcripts (ATIP1, ATIP3 and ATIP4), each showing different tissue distribution. ATIP polypeptides are identical in their carboxy-terminal region carrying four coiled-coil domains. In their amino-terminal portion, ATIP polypeptides exhibit distinct motifs for localisation in the cytosol, nucleus or cell membrane, suggesting that MTUS1 gene products may be involved in a variety of intracellular functions in an AT2-dependent and independent manner. ATIP1 is ubiquitous and highly expressed in the brain. ATIP3 is the major transcript in tissues (prostate, bladder, breast, ovary, colon) corresponding to cancer types with frequent loss of heterozygosity at 8p22. Interestingly, ATIP4 is a brain-specific transcript highly abundant in the cerebellum and fetal brain. High evolutionary conservation of ATIP amino-acid sequences suggests important biological roles for this new family of proteins in tumor suppression and/or brain function.

Mutation analysis of the 8p22 candidate tumor suppressor gene ATIP/MTUS1 in hepatocellular carcinoma.

A high frequency of allelic loss affecting chromosome 8p and a minimal region of deletion at p21-22 have been previously reported in hepatocellular carcinoma (HCC), suggesting that at least one tumor suppressor gene is present in this region. In this study, we assessed whether the angiotensin II AT2 receptor interacting protein (ATIP)/mitochondrial tumor suppressor gene (MTUS1), a gene newly identified at position 8p22, may be a candidate tumor suppressor gene mutated in HCC. We searched for alterations in the 17 coding exons of ATIP/MTUS1 by means of denaturating high-performance liquid chromatography and sequencing, in 51 HCC tumors and 58 cell lines for which loss of heterozygosity status was known. Five major nucleotide substitutions were identified, all located in exons used by the ATIP3 transcript which is the only ATIP transcript variant expressed in liver. These nucleotide variations result in amino-acid substitution or deletion of conserved structural motifs (nuclear localisation signal, polyproline motif, leucine zipper) and also affect exonic splicing enhancer motifs and physiological splice sites, suggesting potential deleterious effects on ATIP3 function and/or expression.

Blood-brain barrier-specific properties of a human adult brain endothelial cell line.

Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT.

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.

Functional endothelin ET B receptors are selectively expressed in human oligodendrogliomas.

Endothelin-1 (ET-1), a vasoactive and mitogenic peptide mainly produced by vascular endothelial cells, may be involved in the progression of several human tumors. Here, we present an immunohistochemical analysis of the expression pattern of ET-1 receptor subtypes (ET(A)-R and ET(B)-R) and a functional study of their potential role in human oligodendrogliomas and oligoastrocytomas. By comparison, we assessed the corresponding expression patterns of glioblastomas. Interestingly, a nuclear localization of ET-1 receptor subtypes (associated or not with a cytoplasmic labeling) was constantly observed in tumor cells from all three glioma types. Moreover, we noted a distinct receptor distribution in the different gliomas: a nuclear expression of ET(B)-R by tumor cells was found to be restricted to oligodendrogliomas and oligoastrocytomas, while a nuclear expression of ET(A)-R was only detected in tumor cells from some glioblastomas. Using primary cultures of oligodendroglial tumor cells, we confirmed the selective expression of nuclear ET(B)-R, together with a plasma membrane expression, and further demonstrated that this receptor was functionally coupled to intracellular signaling pathways known to be involved in cell survival and/or proliferation: extracellular signal-regulated kinase and focal adhesion kinase activation, actin cytoskeleton reorganization. In addition, impairment of ET(B)-R activation in these cells by in vitro treatment with an ET(B)-R-specific antagonist induced cell death. These data point to ET-1 as a possible survival factor for oligodendrogliomas via ET(B)-R activation and suggest that ET(B)-R-specific antagonists might constitute a potential therapeutic alternative for oligodendrogliomas.

Interactions between lymphocytes, macrophages, and central nervous system cells.

Brain has often been considered as an "immunologically privileged organ," not normally accessible to leukocyte traffic, at least in part because of the presence of the blood-brain barrier, constituted by a specialized microvasculature and surrounding astrocytes, which restricts the exchanges between blood and brain. However, more recent studies have revealed that activated leukocytes can cross into the CNS, at very low levels under normal conditions, in much higher numbers during neuropathological disorders like multiple sclerosis or retroviral infection, and, within brain parenchyma, interact with CNS cells. The present review will thus highlight the multidirectional communication network, based on adhesion molecule expression and cytokine production, which appears in such situations between infiltrated leukocytes, brain microvessel endothelial cells, macroglia (including astrocytes and oligodendrocytes), microglial cells and neurons.

Protective effect of the multitarget compound DPH-4 on human SSAO/VAP-1-expressing hCMEC/D3 cells under oxygen-glucose deprivation conditions: an in vitro experimental model of cerebral ischaemia.

BACKGROUND AND PURPOSE: Stroke and Alzheimer's disease (AD) are related pathologies in which the cerebrovascular system is involved. Plasma levels of semicarbazide-sensitive amine oxidase/vascular adhesion protein 1 (SSAO/VAP-1, also known as Primary Amine Oxidase -PrAO) are increased in both stroke and AD patients and contribute to the vascular damage. During inflammation, its enzymatic activity mediates leukocyte recruitment to the injured tissue, inducing damage in the blood-brain barrier (BBB) and neuronal tissue. We hypothesized that by altering cerebrovascular function, SSAO/VAP-1 might play a role in the stroke-AD transition. Therefore, we evaluated the protective effect of the novel multitarget-directed ligand DPH-4, initially designed for AD therapy, on the BBB. EXPERIMENTAL APPROACH: A human microvascular brain endothelial cell line expressing human SSAO/VAP-1 was generated, as the expression of SSAO/VAP-1 is lost in cultured cells. To simulate ischaemic damage, these cells were subjected to oxygen and glucose deprivation (OGD) and re-oxygenation conditions. The protective role of DPH-4 was then evaluated in the presence of methylamine, an SSAO substrate, and/or ß-amyloid (Aß). KEY RESULTS: Under our conditions, DPH-4 protected brain endothelial cells from OGD and re-oxygenation-induced damage, and also decreased SSAO-dependent leukocyte adhesion. DPH-4 was also effective at preventing the damage induced by OGD and re-oxygenation in the presence of Aß as a model of AD pathology. CONCLUSIONS AND IMPLICATIONS: From these results, we concluded that the multitarget compound DPH-4 might be of therapeutic benefit to delay the onset and/or progression of the neurological pathologies associated with stroke and AD, which appear to be linked.

Lack of histamine synthesis and down-regulation of H1 and H2 receptor mRNA levels by dexamethasone in cerebral endothelial cells.

The purpose of this work was to determine whether cerebral endothelial cells have the capacity to synthesize histamine or to express mRNA of receptors that specifically respond to available free histamine. The histamine concentrations and the expression of L-histidine decarboxylase (HDC) and histamine H1 and H2 receptor mRNA, both in adult rat brain and in cultured immortalized RBE4 cerebral endothelial cells, were investigated. In this study endothelial cells were devoid of any kind of detectable histamine production, both in vivo and in the immortalized RBE4 cells in culture. Both the immunostainings for histamine and the in situ hybridizations for HDC were negative, as well as histamine determinations by HPLC, indicating that endothelial cells do not possess the capacity to produce histamine. Also, glucocorticoid (dexamethasone) treatment failed to induce histamine production in the cultured cells. Although the cerebral endothelial cells lack histamine production, a nonsaturable uptake in RBE4 cells is demonstrated. The internalized histamine is detected both in the cytoplasm and in the nucleus, which could indicate a role for histamine as an intracellular messenger. Histamine H1 and H2 receptor mRNA was expressed in RBE4 cells, and glucocorticoid treatment down-regulated the mRNA levels of both H1 and H2 receptors. This mechanism may be involved in glucocorticoid-mediated effects on cerebrovascular permeability and brain edema.