Bifidobacterium breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

BACKGROUND AND AIM: Bifidobacterium breve was the first bacteria isolated in the feces of healthy infants and is a dominant species in the guts of breast-fed infants. Some strains of B. breve have been shown to be effective at relieving intestinal inflammation, but the modes of action have yet to be elucidated. In this study, we investigated the mechanisms of action of B. breve CBT BR3 isolated from South Korean infant feces in relieving colitis in vitro and in vivo. METHODS: Colitis was induced in mice with dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS). Quantitative reverse-transcription polymerase chain reaction, in vitro FITC-dextran flux permeability assay, and aryl hydrocarbon receptor (AhR) luciferase assay are performed using Caco-2 cells and HT29-Lucia™ AhR cells. RESULTS: B. breve CBT BR3 was orally administered. B. breve CBT BR3 improved colitis symptoms in both DSS- and DNBS-induced colitis models. B. breve CBT BR3 increased the number of goblet cells per crypt. B. breve increased the mRNA expressions of Notch, Spdef, Muc5, and Il22. The mRNA expressions of Occludin, which encodes a membrane tight-junction protein, and Foxo3, which encodes a protein related to butyrate metabolism, were also increased in the DSS- and DNBS-induced colitis models. B. breve CBT BR3 protected inflammation-induced epithelial cell permeability and improved goblet cell function by inducing aryl hydrocarbon receptor in vitro. CONCLUSIONS: These results indicate that B. breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

BAFF Inhibition Effectively Suppresses the Development of Anti-HLA.A2 Antibody in the Highly Sensitized Mouse Model.

B cell activating factor (BAFF) is a cytokine that plays a role in the survival, proliferation and differentiation of B cells. We proposed to observe the effects of BAFF inhibition on the humoral immune responses of an allosensitized mouse model using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and were treated with anti-BAFF monoclonal antibody (mAb) (named Sandy-2) or control IgG1 antibody. HLA.A2-specific IgG was reduced in BAFF-inhibited mice compared to the control group (Δ-13.62 vs. Δ27.07, p < 0.05). BAFF inhibition also resulted in increased pre-pro and immature B cell proportions and decreased mature B cells in the bone marrow (p < 0.05 vs. control). In the spleen, an increase in transitional B cells was observed with a significant decrease in marginal and follicular B cells (p < 0.05 vs. control). There was no significant difference in the proportions of long-lived plasma and memory B cells. Microarray analysis showed that 19 gene probes were significantly up- (>2-fold, p < 0.05) or down-regulated (≤2-fold, p < 0.05) in the BAFF-inhibited group. BAFF inhibition successfully reduced alloimmune responses through the reduction in alloantibody production and suppression of B cell differentiation and maturation. Our data suggest that BAFF suppression may serve as a useful target in desensitization therapy.

S100A8 and S100A9 in saliva, blood and gingival crevicular fluid for screening established periodontitis: a cross-sectional study.

BACKGROUND: Periodontitis is one of major oral diseases, which has no consensus on early screening tool. This study aimed to compare the association and screening ability of S100A8 and S100A9 in saliva, blood and gingival crevicular fluid (GCF) for periodontitis status. METHODS: We recruited 149 community Korean adults, 50 no or initial periodontitis (NIPERIO) and 99 established periodontitis (PERIO). Using clinical attachment loss and a panoramic radiograph, stage II-IV of new classification of periodontitis proposed at 2018 was considered cases as PERIO. Enzyme linked immunosorbent assay kit was used to quantify S100A8 and S100A9. T-test, analysis of covariance, Mann-Whitney test and correlation analysis were applied to compare the relationship of S100A8 and S100A9 in saliva, blood, and GCF for periodontitis. Receiver operating characteristic curve was applied for screening ability. RESULTS: Among S100A8 and S100A9 in saliva, blood and GCF, S100A8 in saliva was significantly higher in PERIO than in NIPERIO (p < 0.05). However, S100A8 and S100A9 in GCF were higher in NIPERIO (p < 0.05). The screening ability of salivary S100A8 was 75% for PERIO, while that of GCF S100A8 was 74% for NIPERIO. Salivary S100A8 was positively correlated to blood S100A8 (p < 0.05). CONCLUSION: Salivary S100A8 could be a potential diagnostic marker for established periodontitis and be useful for screening established periodontitis.

Association of toothbrushing and proximal cleaning with periodontal health among Korean adults: Results from Korea National Health and Nutrition Examination Survey in year 2010 and 2012.

AIM: Toothbrushing (TB), dental flossing (DF) and inter-dental brushing (IDB) are regarded as fundamental self-care methods for periodontal health. Few evidences on its effectiveness on periodontal health are available. Hence, this study aimed to evaluate the association of TB, DF, IDB and interaction effect with periodontal health. MATERIALS AND METHODS: The nationally representative 4,766 Korean adults aged 19 years and older were cross-sectionally surveyed in 2010 and 2012. Periodontal health was defined as Community Periodontal Index 1-2 for gingivitis and 3-4 for periodontitis. The information about variables was from interview and blood analyses. Multivariable logistic regression analyses and the interaction effect between TB and proximal cleaning (PC: DF and/or IDB) were applied. RESULTS: Toothbrushing thrice or more per day and DF were associated with a lower prevalence of periodontitis by both 44%, while the preventive fraction of DF on gingivitis was 30%. The preventive fraction of interaction effects between TB thrice or more and PC were 78% for periodontitis and 68% for gingivitis among 40-59 year age group. CONCLUSIONS: Toothbrushing and PC are independently associated with periodontal health. Hence, periodontists should recommend TB thrice or more per day and PC such as DF and IDB to promote periodontal health.

Progressive accumulation of autofluorescent granules in macrophages in rat striatum after systemic 3-nitropropionic acid: a correlative light- and electron-microscopic study.

A variety of tissue biomolecules and intracellular structures are known to be autofluorescent. However, autofluorescent signals in brain tissues often confound analysis of the fluorescent markers used for immunohistochemistry. While investigating tissue and cellular pathologies induced by 3-nitropropionic acid, a mitochondrial toxin selective for striatal neurons, we encountered many autofluorescent signals confined to the lesion core. These structures were excited by blue (wavelength = 488 nm) and yellow-orange (555 nm), but not by red (639 nm) or violet (405 nm) lasers, indicating that this autofluorescence overlaps with the emission spectra of commonly used fluorophores. Almost all of the autofluorescence was localized in activated microglia/macrophages, while reactive astrocytes emitted no detectable autofluorescence. Amoeboid brain macrophages filled with autofluorescent granules revealed very weak expression of the microglial marker, ionized calcium-binding adaptor molecule 1 (Iba1), while activated microglia with evident processes and intense Iba1 immunoreactivity contained scant autofluorescent granules. In addition, immunolabeling with two lysosomal markers, ED1/CD68 and lysosomal-associated membrane protein 1, showed a pattern complementary with autofluorescent signals in activated microglia/macrophages, implying that the autofluorescent structures reside within cytoplasm free of intact lysosomes. A correlative light- and electron-microscopic approach finally revealed the ultrastructural identity of the fluorescent granules, most of which matched to clusters of lipofuscin-like inclusions with varying morphology. Thus, autofluorescence in the damaged brain may reflect the presence of lipofuscin-laden brain macrophages, which should be taken into account when verifying any fluorescent signals that are likely to be correlated with activated microglia/macrophages after brain insults.

Ethanol-dispersed and antibody-conjugated polymer nanofibers for the selective capture and 3-dimensional culture of EpCAM-positive cells.

Electrospun and ethanol-dispersed polystyrene-poly(styrene-co-maleic anhydride) (PS-PSMA) nanofibers (NFs) were used as a platform for the selective capture and three-dimensional culture of EpCAM-positive cells in cell culture medium and whole blood. The NFs were treated with streptavidin to facilitate bond formation between the amino groups of streptavidin and the maleic anhydride groups of the NFs. A biotinylated anti-EpCAM monoclonal antibody (mAb) was attached to the streptavidin-conjugated NFs via the selective binding of streptavidin and biotin. Upon simple mixing and shaking with EpCAM-positive cancer cells in a wide concentration range from 10 to 1000,000 cells per 10mL, the mAb-attached NFs (mAb-NFs) captured the Ep-CAM positive cells in an efficiency of 59%-67% depending on initial cell concentrations, with minor mechanical capture of 14%-36%. Captured cells were directly cultured, forming cell aggregates, in the NF matrix, which ensures the cell proliferation and follow-up analysis. Furthermore, the capture capacity of mAb-NFs was assessed in the presence of whole blood and blood lysates, indicating cluster formation that captured target cells. It is anticipated that the antibody-attached NFs can be employed for the capture and analysis of very rare EpCAM positive circulating cancer cells.

Expression of SOCS2 mRNA and protein in the ischemic core and penumbra after transient focal cerebral ischemia in rats.

The suppressor of cytokine signaling 2 (SOCS2) has been reported to be involved in astroglial reactions and adult neurogenesis in the ischemic hippocampus. To elucidate whether SOCS2 is implicated in the pathophysiology of stroke, we investigate spatiotemporal regulation and identification of cell phenotypes expressing SOCS2 after transient focal cerebral ischemia. Weak hybridization signals for SOCS2 mRNA were constitutively observed in striatal neurons and upregulation of SOCS2 mRNA was induced in association with nestin-positive cells in stroke-lesioned rats. Analysis of the characteristics and phenotypes of SOCS2/nestin double-labeled cells revealed spatial differences between infarct and peri-infarct areas. SOCS2/nestin double-labeled cells in the infarct area were associated with the vasculature and were highly proliferative. In contrast, the double-labeled cells in the peri-infarct area were indeed glial fibrillary acidic protein (GFAP)-positive reactive astrocytes forming the glial scar, although nestin-negative reactive astrocytes also exhibited weak SOCS2 expression. In addition, induction of SOCS2 expression was observed in Iba1-positive cells showing a macrophage-like phenotype with amoeboid morphology; these cells were predominantly localized in the infarct area. In the peri-infarct area, only a small proportion of Iba1-positive cells with the morphology of brain macrophages expressed SOCS2 and most activated stellate microglial cells with thick and short processes exhibited weak or negligible SOCS2 expression. Thus, our results revealed the phenotypic and functional heterogeneity of SOCS2-expressing cells within infarct and peri-infarct areas, suggesting the involvement of SOCS2 in astroglial reactions and activation/recruitment of brain macrophages and its potential role in perivascular progenitors/stem cells after ischemic stroke.

Increased Expression of Slit2 and its Robo Receptors During Astroglial Scar Formation After Transient Focal Cerebral Ischemia in Rats.

Slit2, a secreted glycoprotein, has recently been implicated in the post-ischemic astroglial reaction. The objective of this study was to investigate the temporal changes and cellular localization of Slit2 and its receptors, Robo1, Robo2, and Robo4, in a rat transient focal ischemia model induced by middle cerebral artery occlusion. We used double- and triple-immunolabeling to determine the cell-specific changes in Slit2 and its receptors during a 10-week post-ischemia period. The expression profiles of Slit2 and the Robo receptors shared overlapping expression patterns in sham-operated and ischemic striatum. Constitutive expression of Slit2 and Robo receptors was observed in striatal neurons with weak intensity, whereas in rats reperfused after ischemic insults, these immunoreactivities were increased in reactive astrocytes. Astroglial induction of Slit2 and Robo in the peri-infarct region was distinct on days 7-14 after reperfusion and thereafter increased progressively throughout the 10-week experimental period. Slit2 and Robo were prominently expressed in the perinuclear cytoplasm and main processes of reactive astrocytes forming the astroglial scar. This observation was confirmed by quantification of the mean fluorescence intensity of Slit2 and Robo receptors over reactive astrocytes localized at the edge of the infarct area. However, activated microglia/macrophages in the peri-infarct area were devoid of any specific labeling for Slit2 and Robo. Thus, our data revealed a selective and sustained induction of Slit2 and Robo in astrocytes localized throughout the astroglial scar after ischemic stroke, suggesting that Slit2/Robo signaling participates in glial scar formation and brain remodeling following ischemic injury.

Induction of Krüppel-like factor 4 expression in reactive astrocytes following ischemic injury in vitro and in vivo.

Krüppel-like factor 4 (KLF4) is a transcription factor with diverse and cell type-specific functions and is associated with a variety of pathophysiological processes. Recently, it has been proposed that the regulation of KLF4 is critical to neuronal differentiation and that neural progenitors overexpressing KLF4 take on a glial identity. The present study aimed to determine whether KLF4 is involved in the astroglial reaction induced by ischemia-reperfusion injury in the brain. No specific KLF4 immunoreactivity was observed in resting astrocytes of the control hippocampus, but significant induction was detected in reactive astrocytes preferentially located in the CA1 and dentate hilar regions of the hippocampus following transient forebrain ischemia. Astroglial KLF4 expression was induced in the nuclei and cytoplasm within 3 days of ischemia and persisted for at least 4 weeks. This pattern was reproduced in an in vitro astrogliosis model of rat primary cortical astrocytes exposed to oxygen-glucose deprivation (OGD). Furthermore, immunoblot assay showed that nuclear and cytosolic extracts from cortical astrocytes subjected to OGD had significantly higher levels of KLF4 protein compared to normoxic extracts. Thus, our data demonstrate that KLF4 expression was induced in astroglia by ischemic injury both in vivo and in vitro, suggesting that KLF4 may act as a transcription factor linked to the regulation of the astroglial reaction following ischemic injury.

Innominate artery occlusion: a case study.

Aim of the study: The aim of this case report is to evaluate carotid duplex and hemodynamic patterns in an asymptomatic male patient with innominate artery occlusion. Innominate artery occlusion is a rare clinical entity that can lead to a range of cerebrovascular symptoms, including arm claudication, subclavian steal syndrome, and stroke. The case report emphasizes key learning points in diagnosing innominate artery occlusion using imaging and physiological methods. Case description: A 64-year-old asymptomatic male patient with a history of carotid bruit, hypertension, coronary artery bypass grafting, aortic aneurysm, hyperlipidemia, mild aortic stenosis, long-term tobacco use, and a body mass index of 24 was referred for a carotid ultrasound. Conclusions: Innominate artery occlusion is a rare condition requiring a comprehensive assessment of collateralization before any intervention is attempted. Considering waveform features such as transient end-diastolic flow reversal and tardus parvus, along with brachial pressures and transcranial Doppler, can assist in evaluating the extent of disease.

Deleting autotaxin in LysM+ myeloid cells impairs innate tumor immunity in models of metastatic melanoma.

Autotaxin (ATX) is a lysophospholipase D that generates lysophosphatidic acid (LPA) and regulates cancer metastasis, therapeutic resistance, and tumor immunity. We found that myeloid cells in human melanoma biopsies abundantly express ATX and investigated its role in modulating innate tumor immunity using two models of melanoma metastasis-spontaneous and experimental. Targeted knockout of ATX in LysM+ myeloid cells in mice (LysM-KO) reduced both spontaneous and experimental B16-F10 melanoma metastases by ≥ 50%. Immunoprofiling revealed differences in M2-like alveolar macrophages, neutrophils and regulatory T cells in the metastatic lungs of LysM-WT versus LysM-KO that are model-dependent. These differences extend systemically, with LysM-KO mice bearing experimental metastasis having fewer neutrophils in the spleen than LysM-WT mice. Our results show that (1) LysM+ myeloid cells are an important source of ATX/LPA that promote melanoma metastasis by altering innate tumor immunity, and (2) intratumor and systemic immune profiles vary dynamically during disease progression and are model-dependent.

Single-cell RNA sequencing revealed the role of the Th17 pathway in the development of anti- human leukocyte antigen antibodies in a highly sensitized mouse model.

Background: The aim of this study is to investigate the specific pathway involved in human leukocyte antigen (HLA) sensitization using single-cell RNA-sequencing analysis and an allo-sensitized mouse model developed with an HLA.A2 transgenic mouse. Methods: For sensitization, wild-type C57BL/6 mouse received two skin grafts from C57BL/6-Tg(HLA-A2.1)1Enge/J mouse (allogeneic mouse, ALLO). For syngeneic control (SYN), skin grafts were transferred from C57BL/6 to C57BL/6. We performed single-cell RNA-sequencing analysis on splenocytes isolated from ALLO and SYN and compared the gene expression between them. Results: We generated 9,190 and 8,890 single-cell transcriptomes from ALLO and SYN, respectively. Five major cell types (B cells, T cells, natural killer cells, macrophages, and neutrophils) and their transcriptome data were annotated according to the representative differentially expressed genes of each cell cluster. The percentage of B cells was higher in ALLO than it was in SYN. Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that the highly expressed genes in the B cells from ALLO were mainly associated with antigen processing and presentation pathways, allograft rejection, and the Th17 cell differentiation pathway. Upregulated genes in the T cells of ALLO were involved in the interleukin (IL)-17 signaling pathway. The ratio of Th17 cluster and Treg cluster was increased in the ALLO. On flow cytometry, the percentage of Th17 (IL-17+/CD4+ T) cells was higher and regulatory T cells (FOXP3+/CD4+ T) was lower in the ALLO compared to those in the SYN. Conclusion: Our results indicate that not only the B cell lineage but also the Th17 cells and their cytokine (IL-17) are involved in the sensitization to HLA.

Characterization of nestin expression and vessel association in the ischemic core following focal cerebral ischemia in rats.

The present study aimed to provide a detailed characterization of the cellular phenotypes of nestin-positive cells in a rat model of ischemic stroke. Nestin-positive cells included reactive astrocytes in the peri-infarct region. In the ischemic core, in which astrocytes had virtually disappeared, nestin expression was exclusively associated with the vasculature, including the microvasculature and larger caliber vessels. Induction of nestin expression in the ischemic core occurred by 3 days post-ischemia. Nestin expression continued through at least 28 days post-ischemia but the cellular profiles of nestin-positive cells changed over this period. In the ischemic core at day 3, nestin-positive cells frequently had long processes that ran parallel along the longitudinal axis of the vasculature. These cells were highly proliferative and expressed the transcription factor for neural/glial progenitors, Sox9. Based on their morphological characteristics and on a double-labeling study, most nestin-positive cells were clearly distinguishable from vasculature-associated cells including endothelial cells, smooth muscle cells and microglia/macrophages. Immunoelectron microscopic findings demonstrated that most nestin-positive cells lay in the perivascular space and had macrophage-like features, indicating morphological similarity to perivascular macrophages. Nestin expression was still associated with the vasculature 14 days after ischemia but appeared in fibroblast-like cells. Thus, our data indicated that, in the ischemic core, nestin expression was not limited to a progenitor/stem cell population but was induced in the vasculature-associated cells. These cell types included perivascular macrophages and fibroblast-like cells that appeared to undergo dynamic structural changes. These results suggest that nestin facilitates cellular structural remodeling in response to ischemic injury.

Cross-reactive antibodies to target proteins are dependent upon oligomannose glycosylated epitopes in HTLV-1 associated neurological disease.

Our lab recently identified a cross-reactive antibody response between human T-lymphotropic virus type-1-p24-(gag) (HTLV-1-p24-(gag)) and peroxiredoxin-1 (PrX-1) as potentially contributing to the pathogenesis of HTLV-1 associated neurological disease via molecular mimicry. These targets proteins were glycosylated, yet the glycan side chains immunoreactive with the immunoglobulins were unknown. Using a combination of lectin isolation and serial enzymatic deglycosylation of glycoproteins, we determined that the immunoreactive epitopes contained branched oligomannose side chains. These data suggest that post-translational glycosylation specifically related to oligomannose immunoreactivity to both the infecting and host antigens may contribute to molecular mimicry and be important in the pathogenesis of HTLV-1 associated neurological disease.

Engineered human blood-brain barrier microfluidic model for vascular permeability analyses.

The blood-brain barrier (BBB) greatly restricts the entry of biological and engineered therapeutic molecules into the brain. Due to challenges in translating results from animal models to the clinic, relevant in vitro human BBB models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. This protocol describes an in vitro model of the human BBB self-assembled within microfluidic devices from stem-cell-derived or primary brain endothelial cells, and primary brain pericytes and astrocytes. This protocol requires 1.5 d for device fabrication, 7 d for device culture and up to 5 d for downstream imaging, protein and gene expression analyses. Methodologies to measure the permeability of any molecule in the BBB model, which take 30 min per device, are also included. Compared with standard 2D assays, the BBB model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. These properties, coupled with a functional brain endothelial expression profile and the capability to easily test several repeats with low reagent consumption, make this BBB model highly suitable for widespread use in academic and industrial laboratories.

Prometastatic Effect of ATX Derived from Alveolar Type II Pneumocytes and B16-F10 Melanoma Cells.

Although metastases are the principal cause of cancer-related deaths, the molecular aspects of the role of stromal cells in the establishment of the metastatic niche remain poorly understood. One of the most prevalent sites for cancer metastasis is the lungs. According to recent research, lung stromal cells such as bronchial epithelial cells and resident macrophages secrete autotaxin (ATX), an enzyme with lysophospholipase D activity that promotes cancer progression. In fact, several studies have shown that many cell types in the lung stroma could provide a rich source of ATX in diseases. In the present study, we sought to determine whether ATX derived from alveolar type II epithelial (ATII) pneumocytes could modulate the progression of lung metastasis, which has not been evaluated previously. To accomplish this, we used the B16-F10 syngeneic melanoma model, which readily metastasizes to the lungs when injected intravenously. Because B16-F10 cells express high levels of ATX, we used the CRISPR-Cas9 technology to knock out the ATX gene in B16-F10 cells, eliminating the contribution of tumor-derived ATX in lung metastasis. Next, we used the inducible Cre/loxP system (Sftpc-CreERT2/Enpp2fl/fl) to generate conditional knockout (KO) mice in which ATX is specifically deleted in ATII cells (i.e., Sftpc-KO). Injection of ATX-KO B16-F10 cells into Sftpc-KO or Sftpc-WT control littermates allowed us to investigate the specific contribution of ATII-derived ATX in lung metastasis. We found that targeted KO of ATX in ATII cells significantly reduced the metastatic burden of ATX-KO B16-F10 cells by 30% (unpaired t-test, p = 0.028) compared to Sftpc-WT control mice, suggesting that ATX derived from ATII cells could affect the metastatic progression. We detected upregulated levels of cytokines such as IFNγ (unpaired t-test, p < 0.0001) and TNFα (unpaired t-test, p = 0.0003), which could favor the increase in infiltrating CD8+ T cells observed in the tumor regions of Sftpc-KO mice. Taken together, our results highlight the contribution of host ATII cells as a stromal source of ATX in the progression of melanoma lung metastasis.

Physiologic flow-conditioning limits vascular dysfunction in engineered human capillaries.

Hemodynamics play a central role in the health and disease of the coronary and peripheral vascular systems. Vessel-lining endothelial cells are known mechanosensors, responding to disturbances in flow - with mechanosensitivity hypothesized to change in response to metabolic demands. The health of our smallest microvessels have been lauded as a prognostic marker for cardiovascular health. Yet, despite numerous animal models, studying these small vessels has proved difficult. Microfluidic technologies have allowed a number of 3D vascular models to be developed and used to investigate human vessels. Here, two such systems are employed for examining 1) interstitial flow effects on neo-vessel formation, and 2) the effects of flow-conditioning on vascular remodeling following sustained static culture. Interstitial flow is shown to enhance early vessel formation via significant remodeling of vessels and interconnected tight junctions of the endothelium. In formed vessels, continuous flow maintains a stable vascular diameter and causes significant remodeling, contrasting the continued anti-angiogenic decline of statically cultured vessels. This study is the first to couple complex 3D computational flow distributions and microvessel remodeling from microvessels grown on-chip (exposed to flow or no-flow conditions). Flow-conditioned vessels (WSS < 1Pa for 30 µm vessels) increase endothelial barrier function, result in significant changes in gene expression and reduce reactive oxygen species and anti-angiogenic cytokines. Taken together, these results demonstrate microvessel mechanosensitivity to flow-conditioning, which limits deleterious vessel regression in vitro, and could have implications for future modeling of reperfusion/no-flow conditions.

Osteopontin: correlation with phagocytosis by brain macrophages in a rat model of stroke.

Osteopontin (OPN) is an adhesive glycoprotein linked to a variety of pathophysiological processes. We investigated whether OPN might act as an opsonin in the diseased brain by studying the postischemic expression and localization of OPN mRNA and protein in a rat model of ischemic stroke. In addition, we characterized the subcellular localization of OPN protein in the ischemic brain core. Induction of OPN mRNA occurred in activated microglia/macrophages in the ischemic core on days 3-7 after reperfusion and this was sustained up to day 28, at least. OPN protein was synthesized and secreted by brain macrophages, which first surrounded damaged striatal white matter tracts and then infiltrated into them. Punctate OPN-immunoreactive profiles were scattered throughout the infarction core except in white matter bundles. Electron microscopy showed the localization of OPN protein along the membranes lining what appeared to be the debris of dead neurons. These were located in the extracellular space and within the cytoplasm of brain macrophages, indicating that the OPN protein accumulated selectively on the surface of dead cells, most of which were phagocytosed subsequently by brain macrophages. However, no significant induction of OPN occurred in degenerating striatal white matter tracts or in brain macrophage-engulfed axonic or myelin debris. These data suggest that OPN secreted by brain macrophages in this rat model of stroke might be involved in the phagocytosis of fragmented cell debris and possibly not in the phagocytosis of axonic or myelin debris.

Sprouting angiogenesis under a chemical gradient regulated by interactions with an endothelial monolayer in a microfluidic platform.

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.