PDGFRß-positive cell-mediated post-stroke remodeling of fibronectin and laminin α2 for tissue repair and functional recovery.

Post-stroke intra-infarct repair promotes peri-infarct neural reorganization leading to functional recovery. Herein, we examined the remodeling of extracellular matrix proteins (ECM) that constitute the intact basal membrane after permanent middle cerebral artery occlusion (pMCAO) in mice. Among ECM, collagen type IV remained localized on small vessel walls surrounding CD31-positive endothelial cells within infarct areas. Fibronectin was gradually deposited from peri-infarct areas to the ischemic core, in parallel with the accumulation of PDGFRß-positive cells. Cultured PDGFRß-positive pericytes produced fibronectin, which was enhanced by the treatment with PDGF-BB. Intra-infarct deposition of fibronectin was significantly attenuated in pericyte-deficient Pdgfrb+/-mice. Phagocytic activity of macrophages against myelin debris was significantly enhanced on fibronectin-coated dishes. In contrast, laminin α2, produced by GFAP- and aquaporin 4-positive astrocytes, accumulated strongly in the boundary of peri-infarct areas. Pericyte-conditioned medium increased the expression of laminin α2 in cultured astrocytes, partly through TGFß1. Laminin α2 increased the differentiation of oligodendrocyte precursor cells into oligodendrocytes and the expression of myelin-associated proteins. Peri-infarct deposition of laminin α2 was significantly reduced in Pdgfrb+/-mice, with attenuated oligodendrogenesis in peri-infarct areas. Collectively, intra-infarct PDGFRß-positive cells may orchestrate post-stroke remodeling of key ECM that create optimal environments promoting clearance of myelin debris and peri-infarct oligodendrogenesis.

Deletion of Nox4 enhances remyelination following cuprizone-induced demyelination by increasing phagocytic capacity of microglia and macrophages in mice.

NOX4 is a major reactive oxygen species-producing enzyme that modulates cell stress responses. We here examined the effect of Nox4 deletion on demyelination-remyelination, the most common pathological change in the brain. We used a model of cuprizone (CPZ)-associated demyelination-remyelination in wild-type and Nox4-deficient (Nox4-/- ) mice. While the CPZ-induced demyelination in the corpus callosum after 4 weeks of CPZ intoxication was slightly less pronounced in Nox4-/- mice than that in wild-type mice, remyelination following CPZ withdrawal was significantly enhanced in Nox4-/- mice with an increased accumulation of IBA1-positive microglia/macrophages in the demyelinating corpus callosum. Consistently, locomotor function, as assessed by the beam walking test, was significantly better during the remyelination phase in Nox4-/- mice. Nox4 deletion did not affect autonomous growth of primary-culture oligodendrocyte precursor cells. Although Nox4 expression was higher in cultured macrophages than in microglia, Nox4-/- microglia and macrophages both showed enhanced phagocytic capacity of myelin debris and produced increased amounts of trophic factors upon phagocytosis. The expression of trophic factors was higher, in parallel with the accumulation of IBA1-positive cells, in the corpus callosum in Nox4-/- mice than that in wild-type mice. Nox4 deletion suppressed phagocytosis-induced increase in mitochondrial membrane potential, enhancing phagocytic capacity of macrophages. Treatment with culture medium of Nox4-/- macrophages engulfing myelin debris, but not that of Nox4-/- astrocytes, enhanced cell growth and expression of myelin-associated proteins in cultured oligodendrocyte precursor cells. Collectively, Nox4 deletion promoted remyelination after CPZ-induced demyelination by enhancing microglia/macrophage-mediated clearance of myelin debris and the production of trophic factors leading to oligodendrogenesis.

Low-dose sodium-glucose cotransporter 2 inhibitor ameliorates ischemic brain injury in mice through pericyte protection without glucose-lowering effects.

Antidiabetic sodium-glucose cotransporter 2 (SGLT2) inhibitors have attracted attention for their cardiorenal-protective properties beyond their glucose-lowering effect. However, their benefits in ischemic stroke remain controversial. Here we show the effects of luseogliflozin, a selective SGLT2 inhibitor, in acute ischemic stroke, using a permanent middle cerebral artery occlusion (pMCAO) model in non-diabetic mice. Pretreatment with low-dose luseogliflozin, which does not affect blood glucose levels, significantly attenuated infarct volume, blood-brain barrier disruption, and motor dysfunction after pMCAO. SGLT2 was expressed predominantly in brain pericytes and was upregulated in peri- and intra-infarct areas. Notably, luseogliflozin pretreatment reduced pericyte loss in ischemic areas. In cultured pericytes, luseogliflozin activated AMP-activated protein kinase α and increased mitochondrial transcription factor A expression and number of mitochondria, conferring resistance to oxygen-glucose deprivation. Collectively, pre-stroke inhibition of SGLT2 induces ischemic tolerance in brain pericytes independent of the glucose-lowering effect, contributing to the attenuation of ischemic brain injury.

Reciprocal Interaction Between Pericytes and Macrophage in Poststroke Tissue Repair and Functional Recovery.

BACKGROUND AND PURPOSE: Poststroke tissue repair, comprised of macrophage-mediated clearance of myelin debris and pericyte-mediated fibrotic response within the infarct area, is an important process for functional recovery. Herein, we investigated the reciprocal interaction between pericytes and macrophages during poststroke repair and functional recovery. METHODS: We performed a permanent middle cerebral artery occlusion in both wild-type and pericyte-deficient PDGFRß (platelet-derived growth factor receptor ß) heterozygous knockout (Pdgfrb+/-) mice and compared histological changes and neurological functions between the 2 groups. We also examined the effects of conditioned medium harvested from cultured pericytes, or bone marrow-derived macrophages, on the functions of other cell types. RESULTS: Localization of PDGFRß-positive pericytes and F4/80-positive macrophages was temporally and spatially very similar following permanent middle cerebral artery occlusion. Intrainfarct accumulation of macrophages was significantly attenuated in Pdgfrb+/- mice. Intrainfarct pericytes expressed CCL2 (C-C motif ligand 2) and CSF1 (colony stimulating factor 1), both of which were significantly lower in Pdgfrb+/- mice. Cultured pericytes expressed Ccl2 and Csf1, both of which were significantly increased by PDGF-BB and suppressed by a PDGFRß inhibitor. Pericyte conditioned medium significantly enhanced migration and proliferation of bone marrow-derived macrophages. Poststroke clearance of myelin debris was significantly attenuated in Pdgfrb+/- mice. Pericyte conditioned medium promoted phagocytic activity in bone marrow-derived macrophages, also enhancing both STAT3 (signal transducer and activator of transcription 3) phosphorylation and expression of scavenger receptors, Msr1 and Lrp1. Macrophages processing myelin debris produced trophic factors, enhancing PDGFRß signaling in pericytes leading to the production of ECM (extracellular matrix) proteins and oligodendrogenesis. Functional recovery was significantly attenuated in Pdgfrb+/- mice, parallel with the extent of tissue repair. CONCLUSIONS: A reciprocal interaction between pericytes and macrophages is important for poststroke tissue repair and functional recovery.

Pericyte-Mediated Tissue Repair through PDGFRß Promotes Peri-Infarct Astrogliosis, Oligodendrogenesis, and Functional Recovery after Acute Ischemic Stroke.

Post-stroke functional recovery can occur spontaneously during the subacute phase; however, how post-stroke fibrotic repair affects functional recovery is highly debated. Platelet-derived growth factor receptor ß (PDGFRß)-expressing pericytes are responsible for post-stroke fibrotic repair within infarct areas; therefore, we examined peri-infarct neural reorganization and functional recovery after permanent middle cerebral artery occlusion (pMCAO) using pericyte-deficient Pdgfrb+/- mice. Time-dependent reduction of infarct area sizes, i.e., repair, was significantly impaired in Pdgfrb+/- mice with recovery of cerebral blood flow (CBF) in ischemic areas attenuated by defective leptomeningeal arteriogenesis and intrainfarct angiogenesis. Peri-infarct astrogliosis, accompanied by increased STAT3 phosphorylation, was attenuated in Pdgfrb+/- mice. Pericyte-conditioned medium (PCM), particularly when treated with platelet-derived growth factor subunit B (PDGFB) homodimer (PDGF-BB; PCM/PDGF-BB), activated STAT3 and enhanced the proliferation and activity of cultured astrocytes. Although peri-infarct proliferation of oligodendrocyte (OL) precursor cells (OPCs) was induced promptly after pMCAO regardless of intrainfarct repair, OPC differentiation and remyelination were significantly attenuated in Pdgfrb+/- mice. Consistently, astrocyte-CM (ACM) promoted OPC differentiation and myelination, which were enhanced remarkably by adding PCM/PDGF-BB to the medium. Post-stroke functional recovery correlated well with the extent and process of intrainfarct repair and peri-infarct oligodendrogenesis. Overall, pericyte-mediated intrainfarct fibrotic repair through PDGFRß may promote functional recovery through enhancement of peri-infarct oligodendrogenesis as well as astrogliosis after acute ischemic stroke.

Early initiation of a factor Xa inhibitor can attenuate tissue repair and neurorestoration after middle cerebral artery occlusion.

The timing of anti-coagulation therapy initiation after acute cardioembolic stroke remains controversial. We investigated the effects of post-stroke administration of a factor Xa inhibitor in mice, focusing on tissue repair and functional restoration outcomes. We initiated administration of rivaroxaban, a Xa inhibitor, immediately after permanent distal middle cerebral artery occlusion (pMCAO) in CB-17 mice harboring few leptomeningeal anastomoses at baseline. Rivaroxaban initiated immediately after pMCAO hindered the recovery of blood flow in ischemic areas by inhibiting leptomeningeal anastomosis development, and led to impaired restoration of neurologic functions with less extensive peri-infarct astrogliosis. Within infarct areas, angiogenesis and fibrotic responses were attenuated in rivaroxaban-fed mice. Furthermore, inflammatory responses, including the accumulation of neutrophils and monocytes/macrophages, local secretion of pro-inflammatory cytokines, and breakdown of the blood-brain barrier, were enhanced in infarct areas in mice treated immediately with rivaroxaban following pMCAO. The detrimental effects were not found when rivaroxaban was initiated after transient MCAO or on day 7 after pMCAO. Collectively, early post-stroke initiation of a factor Xa inhibitor may suppress leptomeningeal anastomosis development and blood flow recovery in ischemic areas, thereby resulting in attenuated tissue repair and functional restoration unless occluded large arteries are successfully recanalized.

Subacute Methotrexate Encephalopathy Mimicking Ischemic Stroke With Dynamic Changes on Magnetic Resonance Imaging.

We report a 35-year-old woman who suddenly developed left hemiparesis and dysarthria at 13days after treatment with intrathecal and intravenous methotrexate for intravascular large B cell lymphoma with possible central nervous system infiltration. Seven hours after onset, she developed further right hemiparesis and aphasia. However, the majority of neurologic symptoms disappeared spontaneously and completely by 34hours. We also recorded the dynamic progression and regression of abnormal signals in the bilateral corona radiata on diffusion-weighted imaging, in parallel with neurologic symptoms. The rapid reversal of MR abnormalities and neurologic symptoms allowed us to diagnose methotrexate encephalopathy, and exclude intravascular large B cell lymphoma recurrence and regular brain infarction. The case provides new data on the dynamic changes of abnormal signals on magnetic resonance imaging in methotrexate encephalopathy over a short recovery time.

Incidence of retinal detachment associated with atopic dermatitis in Japan: review of cases from 1992 to 2011.

PURPOSE: The present study aims to investigate the number and characteristics of retinal detachment with atopic dermatitis (AD) in these 20 years, and the number of the first visit AD outpatients in almost the same period. METHODS: A retrospective review of 101 consecutive surgically treated retinal detachments with AD patients from 1992 to 2011 was conducted. Retinal detachments were divided into two groups: eyes operated on from 1992 to 2001 (former AD group, n=63) and eyes operated on from 2002 to 2011 (recent AD group, n=38). We also reviewed the records of the first visit AD outpatients from 1993 to 2011 except 1998. RESULTS: The percentage of bilateral detachment was significantly higher in the former AD group (14/63) than that in the recent AD group (0/38) (P=0.0002). In addition, patients in the recent AD group were significantly older than those in the former AD group (P=0.0084). The annual cases with non-AD retinal detachment remained invariant for 20 years. The ratio of the retinal detachment with AD for the total retinal detachment was significantly lower in the recent (38/847) AD group than that in the former (63/796) AD group (P=0.0038). The number of the first visit AD outpatients linearly decreased in these 19 years (153 cases in 1993 and 65 cases in 2011). CONCLUSION: Our study indicates an apparent decrease in retinal detachment with AD in the recent 10 years, and might suggest the importance of dermatitis control for prevention of retinal detachment with AD.

Immunological parameters in prophylactic sublingual immunotherapy in asymptomatic subjects sensitized to Japanese cedar pollen.

BACKGROUND: This study aims to examine the immunological parameters, focusing IL-10 productivity, in prophylactic sublingual immunotherapy (SLIT) in asymptomatic subjects sensitized to Japanese cedar pollen (JCP). METHODS: This study was conducted as part of a randomized, double-blind, placebo-controlled, multiple center trial, and was performed for two consecutive pollen seasons in 2012 and 2013. The present results were based only on our institution. We recruited 29 participants with specific IgE against JCP of at class 2 and higher levels without history of the pollinosis symptoms at the time of JCP scattering. The SLIT group received standardized JCP extract for five months over the pollen season. We observed and judged development of the symptoms in the pollen season. The percentage of IL-10 producing CD4(+) T (Trl) cells, B cells and monocytes were analyzed by flow cytometry. JCP specific IgE and total IgE were also measured. RESULTS: The ratio of development of cedar pollinosis was significantly lower in the SLIT group compared to the placebo group in 2013. In 2012, the percentage of circulating Tr1 cells and IL-10 producing monocytes significantly increased in the SLIT group. In 2013, the percentage of circulating Tr1 cells and IL-10 producing B cells increased significantly in the SLIT group. The percentage of circulating IL-10 producing monocytes significantly decreased in the placebo group. CONCLUSIONS: Prophylactic SLIT is effective for prevention of the development of pollinosis. Induction of IL-10 producing T cells, B cells and monocytes is an important mechanism of SLIT for prevention of pollinosis in asymptomatic but sensitized subjects.

Single step modified ink staining for Tzanck test: quick detection of herpetic giant cells in Tzanck smear.

Tzanck test has been recently re-evaluated as a method for the diagnosis of herpes virus infection. Giemsa staining for the Tzanck test is time-consuming and laborious. There is a need to develop simple and quick staining methods for bedside diagnosis of this disease. We report a single step and quick method for staining herpes giant cells in Tzanck smears using routinely available inks and physiological saline. A keratinocyte cell line (HaCaT) was cultured on a slide glass and stained with various commercially available blue, blue-black and black inks serially diluted with physiological saline. Clinical smear samples from herpes lesions were also stained with these solutions without specific pretreatment. The nuclei of HaCaT were clearly stained showing high contrast with the cytoplasm using 5% Parker-Quink blue-black ink saline solution. Concentration of ink solution higher or lower than 5% resulted in less contrast. Blue or black inks or other manufacturers' inks can also be used, but staining of the cultured keratinocytes was less clear. Smear of clinical samples from herpes lesions were also stained with 5% ink solution. The nuclei of the multinucleated giant cells were clearly stained, and the sample could be immediately used for microscopic examination. One step staining of Tzanck smear using this diluted ink solution is an inexpensive and a convenient bedside diagnostic tool for the dermatologist.

The role of cytokines/chemokines in the pathogenesis of atopic dermatitis.

Atopic dermatitis (AD) is the most common and relapsing allergic disease of the skin. AD is characterized by a predominant expression of Th2-type cytokines associated with increased cellular infiltration in the skin, elevated circulating levels of IgE and eosinophilia. These findings are positively correlated with interleukin (IL)-4 and IL-13 expression in CD4+ T cells. In AD patients, Th2 cells, eosinophils, mast cells and dendritic cells are markedly increased in the skin lesions. However, Th1 cells are also involved in the development of AD lesions. In fact, Th1 cytokine mRNA expressions including γ-interferon and IL-12 are elevated in the chronic lesions as well as elevated Th2 cytokines in the acute AD lesions. The discovery of Th17 lineage and regulatory T (T(reg)) cells shifted the simple Th1/Th2 balance concept into a 4-way balance system. Th17/22 cells, Foxp3+ T(reg) and IL-10-producing T cells (Tr1) are involved in the mechanism of a local and systemic immunological milieu. In addition, super Th1 cells arranged from Th1 cells in high IL-18 milieu are also involved in the development of mouse AD lesions. Correction of Th2 cytokine predominance by Th1 inducers shows effectiveness in experimental models. However, fine-tuning of the delicate 4-way balance among Th1, Th2, Th17/22 and T(reg) cells is required for the control of AD. Efficacy of some biological agents in AD has been reported. However, further investigations are required to make possible the therapeutic application of biologicals, antigen-specific immunotherapy, non-antigen-specific immunotherapy, antagonists and biological response modifiers in the clinic. These novel approaches may constitute a potential curative therapy for AD.

High-scatter T cells: a reliable biomarker for malignant T cells in cutaneous T-cell lymphoma.

In early-stage cutaneous T-cell lymphoma (CTCL), malignant T cells are confined to skin and are difficult to isolate and discriminate from benign reactive cells. We found that T cells from CTCL skin lesions contained a population of large, high-scatter, activated skin homing T cells not observed in other inflammatory skin diseases. High-scatter T (T(HS)) cells were CD4(+) in CD4(+) mycosis fungoides (MF), CD8(+) in CD8(+) MF, and contained only clonal T cells in patients with identifiable malignant Vß clones. T(HS) cells were present in the blood of patients with leukemic CTCL, absent in patients without blood involvement, and contained only clonal malignant T cells. The presence of clonal T(HS) cells correlated with skin disease in patients followed longitudinally. Clonal T(HS) cells underwent apoptosis in patients clearing on extracorporeal photopheresis but persisted in nonresponsive patients. Benign clonal T-cell proliferations mapped to the normal low-scatter T-cell population. Thus, the malignant T cells in both MF and leukemic CTCL can be conclusively identified by a unique scatter profile. This observation will allow selective study of malignant T cells, can be used to discriminate patients with MF from patients with other inflammatory skin diseases, to detect peripheral blood involvement, and to monitor responses to therapy.

SLIT improves cedar pollinosis by restoring IL-10 production from Tr1 and Monocytes∼IL-10 productivity is critical for becoming allergic∼.

BACKGROUND: Allergen-specific immunotherapy (SIT) is currently used for several allergic disorders and IL-10-producing regulatory T cells (Tr1) induced by SIT suppress allergic reactions. We investigated the relation between IL-10 production and acquiring allergy. METHODS: A prospective study was undertaken to evaluate the effect of SIT on IL-10 production in T cells and other cell fractions in children with pollinosis. In addition, blood samples were collected from non-allergic healthy controls and patients with pollinosis to compare the levels of IL-10 production. PBMC were cultured with pollen peptides or control allergens, and the IL-10 production from monocyte and CD4 T cell was analyzed. RESULTS: Monocytes and CD4 T cells from SIT group of patients produced high levels of IL-10, suggesting that the induction of IL-10 is essential for inducing T cell tolerance. IL-10 production from monocytes and T cells was significantly increased in non-allergic controls compared to patients with pollinosis. This high IL-10 production was observed even when PBMC were stimulated with antigens other than pollen peptides. CONCLUSIONS: IL-10 is critical for induction of specific T cell tolerance, and increased production of IL-10 by monocytes and T cells during inflammatory responses or after SIT may influence effector cells in allergy. Present data implicates that the low productivity of IL-10 by monocytes and T cells is closely related with sensitivity to multiple allergens, and resistance to allergic diseases. Augmentation of constitutive IL-10 production from immune system is a potential therapeutic approach for allergic disorders.

Japanese spotted fever with acute hepatic failure: was it associated with Epstein-Barr virus?

BACKGROUND: An 81-year-old female experiencing high fever, fatigue, and loss of appetite was admitted to our hospital and diagnosed with acute cholecystitis. Her condition did not improve and an eschar and erythema subsequently appeared. We then diagnosed Japanese spotted fever (JSF). She recovered immediately after the administration of minocycline. This case differed from other cases because the patient had a remarkably acute hepatic failure. METHODS: Considering that the present case might be associated with other factors, we performed a repeat polymerase chain reaction (PCR) test on the patient's blood that had been collected on admission and stored. RESULTS: Epstein-Barr virus (EBV) was detected in her blood by PCR. CONCLUSION: We consider this case might be associated with EBV.

Immunohistochemical study of cytokeratin expression in nevus sebaceous.

BACKGROUND: The histogenesis of nevus sebaceous (NS) is unclear. METHODS: To elucidate the histogenesis of NS, cytokeratin (CK) profiles were examined immunohistochemically using 10 anti-keratin antibodies in the three stages of NS. RESULTS: In the first stage, stratified differentiated keratins (CK1 and 10) were reduced, and basal keratin (CK14) was increased in the epidermis and primitive follicular structure (PFS). In the second stage, in addition to reduced CK1 and CK10 expressions and increased CK14 expression, CK17 expression was strongly expressed in the sebaceous ducts in proportion to the development of sebaceous gland. In the third stage, CK14, CK17 and CK19 were expressed in secondary tumors. CK16 was not detected throughout all stages of NS. CONCLUSION: These results suggest that NS is not hyperproliferative but involves hamartomatous differentiation with undifferentiated keratins.

Restoration of peripheral blood T cell repertoire complexity during remission in advanced cutaneous T cell lymphoma.

In advanced stages, cutaneous T cell lymphomas (CTCL) are associated with increased mortality from infections and also increased susceptibility to skin malignancies. In this study, we analyzed the complexity of the peripheral blood T cell repertoire with a sensitive b-variable (BV) complementarity-determining region 3 (CDR3) spectratyping analysis and flow cytometry in three-stage IV CTCL/Sezary syndrome patients who achieved complete clinical remission after therapy. The T cell repertoire of peripheral blood T cells before treatment was profoundly abnormal across multiple BV subfamilies. Following treatment, CDR3 spectratype patterns showed dramatic restoration of normal diversity and complexity. However, absolute CD4 counts across multiple BV families remained low for many months, even after identifiable circulating malignant T cell populations were eliminated. These data suggest that the diversity of the T cell repertoire can be recovered after successful treatment of even advanced CTCL.

Primary cutaneous apocrine carcinoma arising within a congenital nevus: Keratins and filaggrin expression suggesting differentiation into the secretory cells of apocrine glands.

Primary cutaneous apocrine carcinoma (PCAC) is a rare neoplasm of skin appendages. To determine the differentiation of apocrine carcinoma, we studied the expression of epithelial keratins and filaggrin immunohistochemically using 10 anti-keratin antibodies againt keratin (K) 1, 7, 8, 10, 14, 15, 16, 17, 18, 19 and the anti-filaggrin antibody. PCAC demonstrated strong positivity for K7, K8, K18 and K19. These keratins are distributed in secretory cells of normal apocrine glands. The tumor cells were negative for K14 and K17. The two keratins exist in myoepithelial cells in normal apocrine glands. Results suggest that PCAC shows differentiation into secretory cells of apocrine glands, although it does not differentiate into myoepithelial cells. K14 is also known as undifferentiated keratin, whereas K17 is considered to be a hyperproliferative keratin. Absence of the expression of K14 and K17 may reflect an indolent clinical course of PCAC.