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We found that neuropsin, an extracellular matrix serine protease, has a regulatory effect on Schaffer-collateral long-term potentiation (LTP) in the mouse hippocampus. Bath application of 1-170 nM recombinant neuropsin modulated early phase LTP in the Schaffer-collateral pathway with a 'bell-shape' dose-response curve. The maximum enhancing activity (134% of control LTP) was found at approximately 2.5 nM. Bath application of a neutralizing antibody against neuropsin in the hippocampal slice resulted in a marked inhibition of the tetanus-induced early phase of LTP. The in vivo continuous intraventricular infusion of an antisense oligonucleotide against neuropsin significantly reduced the amplitude of the tetanus-induced early phase of LTP in vitro. Neuropsin did not directly change the N-methyl D-aspartate (NMDA) current. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic function on extracellular matrix rather than affecting NMDA receptor-mediated calcium increase.

Background: T H2 cytokines are associated with airway inflammation and hyperreactivity in bronchial asthma, and restoration of the T H1 /T H2 imbalance is a potential avenue for novel therapies. IL-18 is a cytokine secreted by activated macrophages, and it shares some of its biologic activities with IL-12, a typical T H1 -type cytokine. Although IL-18 and IL-12 act on T cells synergistically to induce IFN-γ production, the contribution of IL-18 T H1 /T H2 imbalance and to subsequent asthmatic response has not been elucidated in vivo. Objective: We studied a model of allergic asthma in IL-18–deficient mice to investigate the modulatory role of IL-18 on induction and maintenance of T H2 mucosal immunity. We also have investigated the ability of intraperitoneal instilled IL-18 to reduce T H2 mucosal immunity in IL-18–deficient mice. Methods: IL-18–deficient mice immunized to ovalbumin by means of intraperitoneal injection were challenged 3 times with an aerosol of ovalbumin every second day for 8 days. Recombinant (r)IL-18 was intraperitoneally administered in mice before every first challenge. Mice were analyzed for effects on lung eosinophilia, cytokines, and serum IgE levels. Results: In IL-18–deficient mice, levels of eosinophilia and lung damage were significantly higher than in wild-type C57/BL6 litter mates. Intraperitoneal administration of rIL-18 in deficient mice reduced these antigen-induced changes to levels seen in wild-type mice in association with a decrease in IL-4 in bronchoalveolar lavage fluid and lung tissue. However, administration of rIL-18 did not affect the IFN-γ level and somewhat enhanced the production of IL-5. Notably, reconstitution with rIL-18 increased the numbers of cells staining for Fas ligand, as well as apoptotic cells stained by nick end-labeling in bronchial submucosa infiltrates. Conclusion: These findings indicate that in vivo IL-18 not only inhibited antigen-specific T H2 development but also affected apoptosis through Fas-Fas ligand interactions. These data support a role for IL-18 in the complex pathogenesis of allergic inflammation in which IL-18 limited the development of the local inflammatory response to antigen. (J Allergy Clin Immunol 2000;105:45-53.)

Interleukin 2 receptor is expressed not only on the surface of activated T or B lymphocytes, but also on certain lymphoid malignancies. The receptor is released from the cell membrane as soluble form (sIL-2R). Serum sIL-2R level is a sensitive and quantitative marker of circulating peripheral blood mononuclear cell activation or specific tumor cell growth including non-Hodgkin's lymphoma (NHL). However, the relevance of serum sIL-2R levels relating to clinical outcome in adult patients with NHL remains uncertain. Therefore, we investigated the serial serum sIL-2R levels in 28 untreated patients with NHL to evaluate its correlation with clinical characteristics. High serum sIL-2R level (>1000 U/ml) at diagnosis was associated with a high incidence of treatment failure (p=0.03) and poor overall survival (p=0.057). The serum sIL-2R levels decreased significantly after achieving complete remission (p=0.003). Further larger studies are required to evaluate whether serum sIL-2R level is an independent prognostic factor or not. However, adding this parameter to those already employed in the International Prognostic Index would perhaps provide a better prognostic index for adult patients with NHL.

We evaluated possible prognostic factors just before salvage therapy with vincristine, doxorubicin, and dexamethasone (VAD) for 36 patients with refractory multiple myeloma. The median duration from diagnosis to the first VAD salvage was 14 months (range 2-76 months). Among parameters that have been shown to be associated with poor survival, a high serum lactate dehydrogenase (LDH) level was the sole significant predictor of survival. The median survival of patients with high LDH levels was 4 months, whereas that of patients with low LDH levels was 20 months. A multivariate analysis identified high LDH and high age as independent prognostic factors. More aggressive therapies might be indicated for high-LDH patients with refractory myeloma.

T1/ST2, an orphan receptor with homology with the interleukin (IL)-1 receptor family, is expressed constitutively and stably on the surface of T helper type 2 (Th2) cells, but not on Th1 cells. T1/ST2 is also expressed on mast cells, which are critical for Th2-mediated effector responses. To evaluate whether T1/ST2 is required for Th2 responses and mast cell function, we have generated T1/ST2-deficient (T1/ST2−/−) mice and examined the roles of T1/ST2. Naive CD4+ T cells isolated from T1/ST2−/− mice developed to Th2 cells in response to IL-4 in vitro. T1/ST2−/− mice showed normal Th2 responses after infection with the helminthic parasite Nippostrongylus brasiliensis as well as in the mouse model of allergen-induced airway inflammation. In addition, differentiation and function of bone marrow–derived cultured mast cells were unaffected. These findings demonstrate that T1/ST2 does not play an essential role in development and function of Th2 cells and mast cells.

Background The TH2-type cytokines have been reported to contribute to the asthmatic response. STAT6 has an essential role in IL-4 signalling and in production of TH2 cytokines from T cells and is involved in IgE and IgG 1 responses after nematode infections, indicating that STAT6 has an important role in allergic diseases. Objective In this study we investigated the effects of STAT6 deficiency on allergen-induced airways inflammation in mice. Methods Both ovalbumin (OVA)-sensitized STAT6 deficient (STAT6 -/-) mice and wild-type C57BL/6 mice were challenged with aerosolized OVA. Changes in inflammatory cell infiltration and cytokine levels in lung tissue as well as serum immunoglobulin levels were analysed in OVA-challenged STAT6 -/- and wild-type mice. Results The eosinophilia and lung damage normally resulting from aeroallergen challenge were not seen in STAT6 -/- mice. Expression of TH2 cytokines (IL-4 and IL-5) in the lung tissue as well as IgE and IgG1 responses after OVA challenge were profoundly reduced in STAT6 -/- mice, whereas expression of IFNγ was the same in STAT6 -/- mice and wild-type mice after OVA challenge. Immunocytochemical analysis of T cells showed the infiltration of CD4 + T cells but not CD8 + T cells increased into the lung of wild-type mice after OVA challenge. However, the OVA-exposed STAT6 -/- mice demonstrated the infiltration of both CD4 + T cells and CD8 + T cells with a significant increase in percentage and total number of CD8 + T cells compared with OVA-exposed wild-type mice. Conclusion These results indicate that factors which signal through STAT6 are important regulators of eosinophilia of allergic airway inflammation, regulating TH2-type cytokine production both in CD4 + T cells and CD8 + T cells.

Human leukocyte antigen (HLA)-haploidentical hemopoietic stem cell transplantation (HST) is a promising strategy for patients with hematological malignancies without HLA-identical related or unrela...

The 150-kDa oxygen-regulated protein (ORP150) first was described with reference to the central nervous system in cultured astrocytes subjected to dense hypoxia. Subsequently its transcript was found in macrophages within human aortic atherosclerotic plaques, suggesting a role in protecting cells under hypoxic stress. In a mouse model of permanent focal brain ischemia, we aimed to elucidate the constitutive cellular localization in vivo of ORP150 in the central nervous system as well as the sequential alteration in its mRNA and protein expression during this severe ischemic insult. Immunohistochemical study demonstrated that ORP150 protein normally is present predominantly in neurons. The 78-kDa glucose-regulated protein, which is another well-known stress protein retained in the endoplasmic reticulum, also was stained in neurons. During the first 3 h after ischemia, ORP150 antigenicity was markedly enhanced in severely damaged neurons, while the amount of the glucose-regulated protein was decreased. Preceding this change, orp150 mRNA was selectively induced in neurons undergoing postischemic cytoskeletal proteolysis, as early as 1 h after middle cerebral artery occlusion. These results indicated that ORP150 might be regulated by transcriptional level as for many stress proteins, but unlike previously described other stress proteins it was translated in the center of ischemic lesions despite nearly complete energy depletion. In this paper, the biological potentials of ORP150 protein in the setting of brain ischemia in vivo will also be discussed.

Fungal infection is a serious complication in immunocompromised patients, especially those with neutropenia. Itraconazole (ITZ) is expected to be an effective prophylactic agent for fungal infection because it has more activity against Aspergillus species than fluconazole and it is less toxic than amphotericin-B. However, ITZ is available only as an oral capsule, the absorption of which is thought to depend on the presence of acid in the stomach. In this study, the effect of famotidine, an H2-blocker, on the absorption of ITZ was investigated. Patients undergoing chemotherapy for hematological malignancies were enrolled. To minimize the effect of famotidine, the time of ITZ intake was different from that of famotidine intake. The plasma concentrations of ITZ with or without taking famotidine were determined just before and 4 h after ITZ intake. Mean trough and peak concentrations of ITZ without famotidine were 332 ng/ml and 476 ng/ml, respectively. When famotidine was co-administered, the concentrations decreased to 204 ng/ml and 315 ng/ml, respectively. Statistical analyses revealed significant differences between trough concentrations in the presence and absence of famotidine (p = 0.008). There was also a clear tendency toward higher peak concentrations in the plasma concentrations with famotidine (p = 0.06). These findings suggest that famotidine decreases the plasma concentration of ITZ in patients undergoing chemotherapy. Close monitoring of the plasma concentration of ITZ and dose adjustment are required for efficient prophylaxis.

To evaluate the reversibility of neural function in the brainstem following ischemia, we investigated the effect of transient brainstem ischemia on the brainstem auditory evoked potential in gerbils. Brainstem ischemia was produced by bilateral extracranial occlusion of vertebral arteries. Local cerebral blood flow was measured by quantitative autoradiography after 5 min of ischemia and was reduced to less than 3 ml/100 g per min in the pons and lower midbrain, indicating severe and reproducible brainstem ischemia. During brainstem ischemia, brainstem auditory evoked potential waveforms disappeared completely. After a brief ischemic insult (5 min), all four brainstem auditory evoked potential components recovered to normal. After longer ischemic insults (10-30 min), brainstem auditory evoked potential components never recovered to normal. Microtubule-associated protein 2 immunoreactivity revealed differential vulnerability of the acoustic relay nuclei in the brainstem. Neurons in the lateral lemniscus were most vulnerable, followed in order by neurons in the trapezoid body, the superior olive and the cochlear nucleus. We also demonstrated a close relationship between the reversibility of ischemia-induced changes on brainstem auditory evoked potential and ischemic lesions of these relay nuclei. These data may be useful for evaluating the therapeutic window of thrombolytic therapy during acute vertebrobasilar occlusion.

Trophic coupling between cerebral endothelium and their neighboring cells is required for the development and maintenance of blood-brain barrier (BBB) function. Here we report that oligodendrocyte precursor cells (OPCs) secrete soluble factor TGF-β1 to support BBB integrity. Firstly, we prepared conditioned media from OPC cultures and added them to cerebral endothelial cultures. Our pharmacological experiments showed that OPC-conditioned media increased expressions of tight-junction proteins and decreased in vitro BBB permeability by activating TGB-β-receptor-MEK/ERK signaling pathway. Secondly, our immuno-electron microscopic observation revealed that in neonatal mouse brains, OPCs attach to cerebral endothelial cells via basal lamina. And finally, we developed a novel transgenic mouse line that TGF-β1 is knocked down specifically in OPCs. Neonates of these OPC-specific TGF-β1 deficient mice (OPC-specific TGF-β1 partial KO mice: PdgfraCre/Tgfb1flox/wt mice or OPC-specific TGF-β1 total KO mice: PdgfraCre/Tgfb1flox/flox mice) exhibited cerebral hemorrhage and loss of BBB function. Taken together, our current study demonstrates that OPCs increase BBB tightness by upregulating tight junction proteins via TGF-β signaling. Although astrocytes and pericytes are well-known regulators of BBB maturation and maintenance, these findings indicate that OPCs also play a pivotal role in promoting BBB integrity.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of approximately 70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with these in vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes.

To assess the role of tumor necrosis factor-alpha (TNF-alpha) in modulating the process of cerebral ischemic injury, we identified TNF-alpha-producing cells and studied the time course of TNF-alpha expression. Immunoreactivity for TNF-alpha appeared in white matter of the mouse hippocampus as early as 1.5 h following a 30-min global ischemic insult. Double staining for TNF-alpha and glial fibrillary acidic protein (GFAP) suggested that the TNF-alpha-positive cells are most likely microglia, not astrocytes. TNF-alpha immunostaining decreased at 6 and 24 h but increased again at 3 days, when pyramidal neurons showed degeneration. Adjacent-section staining for microglia and double staining with GFAP suggested that TNF-alpha-positive cells in the pyramidal cell layer were microglia and those in the white matter were astrocytes. By 5 days TNF-alpha immunostaining disappeared from these glial cells, while a number of microglia were accumulated in the degenerated hippocampal pyramidal layer. Pyramidal neurons never expressed TNF-alpha immunoreactivity. Western blotting confirmed biphasic TNF-alpha expression. Our findings suggest that early production of TNF-alpha by microglia may activate a cytokine network in post-ischemic brain resulting in TNF-alpha synthesis by astrocytes.

We read with interest a recent article by Ringelstein et al.1 The authors described the results of a phase 2B clinical trial of granulocyte colony-stimulating factor (G-CSF) for patients with stroke, which did not result in any beneficial effects. The authors concluded that the failure of this clinical trial was mainly attributable to the problem of translating findings from the animal laboratory to patients with clinical stroke and thus raised the question, “Are rodent models questionable for predicting stroke drug efficacy in humans?” However, we think that the negative results of this clinical trial had been predictable from previous results of basic experiments with animal models. The clinical trial was designed …

CD25 expression in follicular lymphoma (FL) has not yet been investigated. Eighty-five patients with newly diagnosed FL were retrospectively evaluated. On two-color flow cytometric analysis, CD25 was detected on CD19 + and CD20 + lymphoma cells. CD25 expression in FL tended to be higher than in reactive lymphadenopathy, but was lower than in diffuse large B-cell lymphoma. Patients with CD25 + FL (n = 12) showed clinical features of elevated soluble interleukin-2 receptor (IL-2R) levels, B symptoms and an advanced age compared with CD25 - FL (n = 73). The overall response rate (ORR), progression-free survival (PFS) and overall survival (OS) in patients with CD25 + FL were significantly inferior to those with CD25 - FL (ORR, 60 vs. 93%; 2-year PFS, 32 vs. 80.3%; 6-year OS, 47.4 vs. 85.9%, respectively). Multivariate analysis demonstrated that CD25 positivity is an independent prognostic factor for PFS and OS in FL. CD25 + FL may constitute a distinct subgroup associated with aggressiveness and an inferior prognosis.

The divalent cation zinc has been reported to possess several physiological properties such as blocking apoptotic cell death through an inhibitory effect on Ca(2+)-Mg2+ endonuclease activity, or modulating the neurotoxicity via glutamate receptor subtypes. In the present study, we investigated the effect of peripherally injected zinc on delayed neuronal death seen in the hippocampus after transient global ischemia, in order to elucidate a possible beneficial role on zinc in ischemic neuronal cell death. Forty-five adult Mongolian gerbils of both sexes underwent transient bilateral clipping of the common carotid arteries for 3 min. In the pretreated animals, ZnCl2 (20 mg/kg) was injected subcutaneously once, 1 h before ischemia (superacute group; n = 6) or twice at 24 and 48 h before ischemia (subacute group; n = 14). Histological survey was carried out 3 days later by in situ DNA fragmentation method and 4 days later by hematoxylin-eosin staining by semiquantatively counting dead neurons in the CA1 sector. Subacute zinc pre-administration significantly reduced the nuclear damage and subsequent neuronal death; however, superacutely pre-administered zinc did not protect hippocampal neurons against ischemia but it did not aggravate the effect of ischemia, either. The present study suggested that transfer of exogenous zinc into the intracellular space is required for neuroprotection, presumably via the anti-endonuclease activity.

To assess whether ischemia could induce GAP-43 mRNA expression, we performed in situ hybridization in gerbil brains that had been subjected to 5 min of global ischemia. In control dentate granule cells, little hybridization was detected in contrast to the intense signal generated by pyramidal neurons of the adult hippocampal formation. After ischemia, we detected a robust GAP-43 signal over hippocampal granule cells at 3 h of reperfusion, persisting through 7 days, and disappearing by 14 days. This demonstrated GAP-43 gene induction after ischemia, and suggests that GAP-43 may be involved in reactive events, including fiber sprouting and synaptic reorganization, that follow ischemia.

The present investigation reveals many cortical neurons immunopositive for M35, the monoclonal antibody raised against purified muscarinic acetylcholine receptor (mAChR) proteins, in the early postnatal rat brain. The ontogeny of mAChR expression, exemplified on the parietal neocortex, was studied in a series of rat pups from postnatal days (PD) 1, 3, 7, 14 and 21. Immunoprecipitation in the parietal somatosensory cortex was manifest in the population of pyramidal neurons during postnatal development. In particular during the early postnatal ages, until 2 weeks after birth, M35 immunoreactivity (M35-ir) was present in all neuronal compartments, indicating transportation of mAChR protein in axonal and dendritic processes as observed in light and electron microscopic analysis. The immunoprecipitation in the apical dendrites yielded dense labeling in layer 1 where the distal processes of the pyramidal dendrites branched extensively forming a plexus that intermingled with horizontal fibers in this superficial layer. At PD21, immunolabeling in layer 1 and in axons of pyramidal cells was reduced compared to earlier ages suggesting a transient expression of mAChRs in these neuronal structures. The development of M35-ir in the cortex appeared to antedate that of its cholinergic afferentation as indicated by AChE histochemical study.

砂ネズミ一過性脳虚血モデルを用い, 遅発性神経細胞死の過程におけるα-amino-3-hydroxy-5-methyl-4-isoxazolepro pionic acid (AMPA) 型グルタミン酸レセプターサブユニットのmRNAと蛋白の双方の変動を観察し, 細胞死のメカニズムとの関連を検討した.海馬CA1領域では5分虚血後1日目にGluR1 mRNA, GluR3 mRNAの顕著な発現の低下がみられたがGluR2m RNAの低下は軽度であった.GluR1-3蛋白の虚血後の変化はCA1領域で1日目の免疫陽性構造の局在の変化として認められた.GluR4は錐体細胞よりも虚血に対して抵抗性のあるインターニューロンに主に認められた.以上の結果より, 虚血後AMPAレセプターはdown-regulationを受けることが示唆され, さらに虚血負荷後一過性にGluR2優位のサブユニット構成変化が生じていることが示された.これはCa2切細胞内流入機構の虚血後変化を意味しており, 遅発性神経細胞死の機序の一つを説明するものと考えられた.

脳虚血病態におけるNMDA型グルタミン酸レセプターの関与を検討する目的で, 砂ネズミ一過性脳虚血モデルを用い, 虚血負荷後経時的に海馬のNMDA型グルタミン酸レセプター (NMDAR1) mRNAの変動をin situ hybridization histochemistryにて検討した.遅発性神経細胞死をもたらす5分間の前脳虚血負荷後, 海馬CA1錐体細胞でのNMDARIm RNAシグナルは1日目から発現の低下がみられ, グリア細胞では逆に発現が充進していた.CA1錐体細胞脱落後はおもにグリア細胞での発現のみが観察された.2分間虚血負荷後には神経細胞におけるNMDARI mRNA発現には変化がみられずグリア細胞でのみ発現の充進がみられ, これは負荷後1週間持続して認められた.このことは致死的負荷では神経細胞のNMDAレセプターのdown regulationを示唆する.また遅発性神経細胞死, 虚血耐性獲得双方の過程にグリア細胞でのNMDAレセプターの発現が大きな影響をもたらすことが示唆された.

Forty-eight patients with gastrointestinal (GI) tract B-cell lymphoma (BCL) were analyzed retrospectively. The diagnosis was based on the histological examination of specimens obtained by endoscopic biopsy. Before the diagnosis was made, single-color flow cytometry was performed to analyze the expression of light chains and B-cell antigens including CD10 in the specimens. Restricted light chain (RLC) expression, a marker of B-cell clonality, was defined as κ and λ ratios of either more than 3.0 or less than 0.5. The specimens from 30 patients (62.5%) showed RLC expression. No RLC expression or RLC expression not examined was divided into two groups : those showing CD10 positivity in more than 20% of cells (4 patients, 8.3%) and those showing no positivity (14 patients, 29.2%). The cell number analyzed in the latter group was significantly smaller than that in the other two groups. Abnormal karyotypes were found in the specimens from 8 patients (16.7%). These results indicate that the flow cytometric analysis of endoscopic biopsy specimens is useful when BCL is suspected if an adequate number of cells are obtained.

CD25 (interluekin-2 receptor) expression in diffuse large B-cell lymphoma (DLBCL) cells has been not examined. To characterize CD25(+) DLBCL, 123 patients, who were newly diagnosed with DLBCL, were analyzed by single-color flow cytometry (FCM). CD25-positivity was significantly higher in DLBCL patients (n = 123; mean ± SD, 27.8 ± 30.6%) than in those with reactive lymphadenopathy (n = 16; mean ± SD, 8.6 ± 4.3%) and follicular lymphoma (n = 60; mean ± SD, 12.7 ± 12.4%). By two-color FCM, CD25/CD19 or CD25/CD20 dual positivity in DLBCL patients was shown: mean ± SD, 63.7 ± 25.5% (n = 13) and 55.0 ± 28.1% (n = 14), respectively. Eighty-two percent of the patients with DLBCL received rituximab combined with CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. A cut-off value of 60% with CD25-positivity clearly divided patients with DLBCL into two groups: CD25-high or CD25-low DLBCL. Although clinical and immunophenotypic features were not significantly different in both groups, the former showed a significantly poorer response and more inferior progression-free survival than the latter. CD25 may be a new prognostic marker and could be a therapeutic target in DLBCL.

In our facility, three patients developed tacrolimus (TAC)-induced renal dysfunction after allogeneic hemopoietic stem cell transplantation, although trough levels of TAC were within therapeutic ranges. They received an oral agent of slow-release TAC once a day instead of a regular form oral TAC twice a day. Following treatment with the prolonged-release agent, serum creatinine levels decreased and graft-versus-host disease (GVHD) did not occur. Use of this slow-release formulation may avoid toxic peak concentrations of TAC without the development of GVHD.

Human mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populations.Given these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD model.Our study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD.Our results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.

Stroke is one of the major causes of death and disability in developed countries. The central nervous system (CNS) is known for its limited reparative capacity, but several studies demonstrated that the CNS has some reparative potential and cerebral ischemia is followed by activation of endogenous neurogenesis (Nakatomi et al., 2002; Taguchi et al., 2004). It is well-known that new neurons are continuously generated in specific brain regions such as the subventricular zones (SVZ) (Alvarez-Buylla et al., 2002) and the subgranular zone within the dentate gyrus of the hippocampus (SGZ) (Kuhn et al., 1996). Although adult cerebral cortical neurogenesis remains controversial, accumulating evidence has shown that under pathological conditions, new neurons are generated in the adult mammalian cerebral cortex (Magavi et al., 2000; Jiang et al., 2001; Jin et al., 2006; Yang et al., 2007). This suggests that neural stem/progenitor cells (NSPCs) can be activated in the cortex by brain injury such as ischemic stroke. In support of this notion, we demonstrated that NSPCs develop in the poststroke area of the cortex in the adult murine (Nakagomi et al., 2009a; Nakagomi et al., 2009b; Nakano-Doi et al., 2010; Saino et al., 2010) and human brain (Nakayama et al., 2010), and we referred to these as ischemia/injury-induced NSPCs (iNSPCs). These cells express markers of NSPCs, such as nestin and Sox2. They also form neurospheres that have the capacity for self-renewal, and differentiate into electrophysiologically functional neurons, astrocytes, and myelin-producing oligodendrocytes (Nakagomi et al., 2009a; Nakagomi et al., 2009b; Nakano-Doi et al., 2010; Clausen et al., 2011). In addition, we demonstrated that iNSPCs originate, at least in part, from within the cerebral cortex, but not from SVZ cells (Nakagomi et al., 2009b). However, the detailed origin and identity of the iNSPCs remains unclear. In this chapter, we introduce the characterization and possible origin of iNSPCs based on our reports and recent viewpoint, and compare them to other previously reported types of CNS stem/progenitor cells, including SVZ astrocytes (Doetsch et al., 1999), ependymal cells (Moreno-Manzano et al., 2009), reactive astrocytes (Shimada et al., 2010), resident glia (Zawadzka et al., 2010), and oligodendrocyte precursor cells (OPCs) (Kondo et al., 2000). We also refer to the possible cortical neurogenesis by iNSPCs and to the therapeutic potential of iNSPC transplantation in stroke patients.

Fas antigen mRNA induction in the brain was examined using a transient global cerebral ischemia model in BALB/C mice. Northern blot analysis revealed little Fas antigen mRNA expression in the brains of sham-operated mice. A marked induction of Fas mRNA expression was detected in the brains of mice 6 h after 30 min of cerebral ischemia. These results suggest a possible apoptotic mechanism for cell death, mediated by the Fas antigen, in postischemic brain.

A new gerbil model of hindbrain ischemia was induced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Carbon black studies, performed at 5 min after occlusion, revealed that the pons-medulla oblongata, and the cerebellum were quite ischemic in all animals. Cardiovascular changes in mean arterial blood pressure (MABP) and heart rate were recorded until 30 min after occlusion, and revealed that the typical cerebral ischemic response (i.e., abrupt increase in MABP, bradycardia, and apnea) was elicited in all animals (n = 10). Thirty minutes after occlusion, animals (n = 4) were decapitated and immersion-fixed. Brain sections were stained with hematoxylin-eosin (HE) and also immunostained for microtubule-associated protein 2 in order to evaluate ischemic neuronal damage from 30 min of ischemia. By HE staining, ischemic lesions were detected bilaterally in the oculomotor, the trigeminal motor, the lateral vestibular, and the cerebellar interpositus nucleus. In addition, immunostaining revealed ischemic lesions in several other hindbrain areas. In conclusion, we could successfully establish a new gerbil model of hindbrain ischemia. Carbon black perfusion and hemodynamic studies revealed that severe and reproducible hindbrain ischemia was produced. By histopathological examination, we could also clearly demonstrate symmetrical ischemic lesions in several hindbrain areas.

In the adult mammalian brain, new neurons are continuously generated at the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. In the cerebral cortex, though the generation of new neurons is rarely observed in physiological situations, induction of neurogenesis has been shown under pathological conditions, such as after cerebral ischemia. In this chapter, we introduce the injury-induced neural stem/progenitor cells in the cerebral cortex and its potential for functional recovery. We also refer to the therapeutic potential of exogenous neuronal stem cell transplantation and discuss the future of cell-based therapy for stroke patients.

Transplantation with cryopreserved allogeneic peripheral blood stem cells (PBSCs) from related donors is widely conducted in Japan. To freeze PBSCs, a solution containing dimethyl sulfoxide (DMSO), which can have various adverse effects, is added. DMSO-depleted allogeneic PBSCs were transplanted into 21 patients. The cryoprotectant was manually removed from thawed PBSCs and the cells were mixed with a solution containing citrate dextrose as an anticoagulant and RPMI-1640 medium. DMSO-depleted PBSCs were immediately infused into patients subjected to conditioning. Infusion-related adverse effects were only observed in three patients. The median neutrophil recovery (⩾0·5×10(9)/l) and platelet recovery (⩾20×10(9)/l) were 13·0 and 14·0 days, respectively. Only one patient with mixed-lineage leukemia in non-complete remission did not show engraftment, likely due to a second transplantation and a two-antigen disparity in human leukocyte antigen system A. The results suggest the removal of DMSO from thawed PBSCs to be safe and useful for transplantation.

To assess the role of Cu-Zn superoxide dismutase (CuZnSOD) in regulating cellular antioxidant defenses, we studied the induction of CuZnSOD mRNA by an in situ hybridization technique and of CuZnSOD protein by an immunocytochemical method in the gerbil hippocampus following 5 min of transient global ischemia. For hybridization, we synthesized 48-mer oligonucleotide (base 465–512) complementary to rat CuZnSOD mRNA. Northern blot analysis showed hybridization to a single band of molecular weight 0.65 kb. After 5 min of ischemia, the signal became stronger at 3 and 24 h and returned to the control level 3 days later. In situ hybridization histochemistry revealed an increase in labeling throughout the hippocampus, especially in the granular layer 3 h following ischemia. The increase was prolonged only in the CA1 pyramidal layer after 24 h and was eliminated within 3 days or later. Conversely, analysis by Western blotting revealed that the insult produced few effects on the induction of CuZnSOD protein. Immunocytochemistry for CuZnSOD revealed a reduced immunostaining in the CA1 pyramidal layer at 24 h of recirculation when the persistent expression of CuZnSOD mRNA was shown in the same area. Our findings suggest that the expression of endogenous CuZnSOD is temporarily stimulated by an ischemic insult without increasing the protein level. The prolonged increase in mRNA and the decrease in the protein of CuZnSOD in the CA, neurons seem to imply an important role of the endogenous antioxidant enzyme that protects against the detrimental effects of superoxide radicals on delayed neuronal death.

We report on a case of severe renal failure in a 61-year-old female with multiple myeloma (MM). Two months prior to admission, the patient was diagnosed to have anemia and progressive renal failure associated with urinary Bence Jones protein and was referred to our hospital. A bone marrow biopsy revealed 40% plasma cells with κ light chain restriction. Thus, she was considered to have MM. A renal biopsy revealed neoplastic plasma cell infiltration within the kidney, moderate interstitial fibrosis, tubular atrophy, and punctate, electron-dense material along the peripheral capillary walls, tubular basement membrane, and in the interstitium of the kidney. This suggested that a combination of compression of the tubules and the microvasculature by the infiltrative process, and local light chain deposition-mediated tissue damage might be implicated in the development of renal failure in this patient. Despite a remission of bone marrow plasmacytosis with a bortezomib-based regimen, her renal function gradually deteriorated and a periodic hemodialysis program was finally required. Although the clinical impact of the direct kidney infiltration of neoplastic plasma cells on the longitudinal changes in renal function remains to be delineated, it is reasonable to consider that the infiltration of neoplastic plasma cells associated with local light chain depositions may result in irreversible renal injuries. Obviously, further studies and accumulation of additional experience with renal biopsy are required to better determine the precise and prognostic relationship between renal outcome and morphological alterations among MM patients with varying degrees of renal impairment.

Acute inflammation in the poststroke period exacerbates neuronal damage and stimulates reparative mechanisms, including neurogenesis. However, only a small fraction of neural stem/progenitor cells survives. In this report, by using a highly reproducible model of cortical infarction in SCID mice, we examined the effects of immunodeficiency on reduction of brain injury, survival of neural stem/progenitor cells, and functional recovery. Subsequently, the contribution of T lymphocytes to neurogenesis was evaluated in mice depleted for each subset of T lymphocyte. SCID mice revealed the reduced apoptosis and enhanced proliferation of neural stem/progenitor cells induced by cerebral cortex after stroke compared with the immunocompetent wild-type mice. Removal of T lymphocytes, especially the CD4+ T-cell population, enhanced generation of neural stem/progenitor cells, followed by accelerated functional recovery. In contrast, removal of CD25+ T cells, a cell population including regulatory T lymphocytes, impaired functional recovery through, at least in part, suppression of neurogenesis. Our findings demonstrate a key role of T lymphocytes in regulation of poststroke neurogenesis and indicate a potential novel strategy for cell therapy in repair of the central nervous system. © 2010 Wiley-Liss, Inc.

We propose the peroxidase-catalyzed simultaneous conjugation and hydrogelation of polysaccharide and protein derivatives, each possessing phenolic hydroxyl (Ph) moieties, as a novel route for obtaining protein-polysaccharide conjugate hydrogels. We used alginate, gelatin, and albumin derivatives bearing Ph moieties (Alg-Ph, Gela-Ph, and Alb-Ph) to demonstrate the feasibility. The gelation time of conjugate gels decreased with decreasing H(2)O(2) concentration and with increasing horseradish peroxidase concentration. Gelation time was controllable from a few seconds to 6 min. The repulsion force detected at 40% compression of a conjugate gel obtained from a mixture of Alg-Ph and Gela-Ph at 1.0% (w/v), respectively, was more than 2.8 times larger than that detected for gels produced from 3.0% (w/v) Gela-Ph or 2.0% (w/v) Alg-Ph alone. Cell adhesiveness of gels was tunable by changing the type of protein derivative. A gel from Gela-Ph and Alg-Ph showed higher cell adhesiveness than Alg-Ph gel, but a gel produced from Alb-Ph and Alg-Ph showed a lower cell adhesiveness than Alg-Ph gel. The conjugate gel was degradable by degrading alginate molecules using the nonproteolytic enzyme alginate lyase. The tunable gelation, mechanical properties, and cell adhesiveness of polysaccharide-protein conjugate hydrogels obtained through peroxidase-catalyzed gelation indicates great potential for a wide range of applications, such as scaffolds for tissue engineering and carriers for drug delivery system.

Increasing evidence points to accelerated neurogenesis after stroke, and support of such endogenous neurogenesis has been shown to improve stroke outcome in experimental animal models. The present study analyses post-stroke cerebral cortex after cardiogenic embolism in autoptic human brain. Induction of nestin- and musashi-1-positive cells, potential neural stem/progenitor cells, was observed at the site of ischemic lesions from day 1 after stroke. These two cell populations were present at distinct locations and displayed different temporal profiles of marker expression. However, no surviving differentiated mature neural cells were observed by 90 days after stroke in the previously ischemic region. Consistent with recent reports of neurogenesis in the cerebral cortex after induction of stroke in rodent models, the present current data indicate the presence of a regional regenerative response in human cerebral cortex. Furthermore, observations underline the potential importance of supporting survival and differentiation of endogenous neural stem/progenitor cells in post-stroke human brain.

Deferasirox (DFX) is a newly developed oral iron chelator that enables effective chelation with once daily administration. We describe here a case of transfusional-iron overloaded patient who experienced hematopoietic recovery after DFX administration. A 75-year-old woman with iron overload, who had been diagnosed with MDS (RCMD) and had received a transfusion of red blood cells and platelets regularly for 3 years, enrolled in the phase I clinical trial of ICL670 (DFX) in Japan. DFX administration steadily decreased her serum ferritin levels and chelated overloaded iron effectively. Interestingly, a year after initiation of the trial, she needed fewer blood transfusions, and no more transfusions after the 17th month of the trial. Even after suspending transfusions, her hemoglobin level and platelet count increased continuously, and she now has stable disease without blood transfusions. She has not received any specific treatment for MDS during this period. Examination of the bone marrow aspirates in the 35th month revealed dysplastic cells, indicating no remarkable change in the state of MDS. This case suggests that excess iron hampers hematopoiesis and that adequate iron chelation may improve hematological data in some iron-overloaded patients.

The beneficial effects of angiotensin II type 1 (AT1) receptor blockers (ARB) in cerebrovascular disease have been shown in clinical trials. However, the effects of ARBs vary based on their unique pharmacologic properties. In this study, we focused on telmisartan, a fat-soluble ARB with selective peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonist activity, and investigated its effects on ischemic injury in cerebral vasculature using murine models of both transient and permanent focal ischemia. Analysis by triphenyltetrazolium-staining revealed that pre-treatment of mice with telmisartan reduced stroke volume 72 h after the transient ischemic insult in a dose-dependent manner, though such treatment did not reduce stroke volume due to permanent ischemia. Transient ischemia induced pro-inflammatory adhesion molecules, such as ICAM-1 and P-selectin in the ischemic region, and treatment with telmisartan diminished the expression of these adhesion molecules with diminished infiltration of inflammatory cells. The beneficial effect of telmisartan was attenuated, in part, by administration of a PPAR gamma antagonist. Treatment with valsartan (an ARB without PPAR gamma agonist activity) also decreased ischemic injury after transient ischemia, though to a lesser extent than telmisartan. Our findings indicate that telmisartan has a beneficial effect in a murine model of ischemia/reperfusion injury through blockade of AT1 receptors, and, in addition, due to a positive effect via its specific anti-inflammatory PPAR gamma agonist activity.

Objective The progression of myelodysplastic syndrome to acute myeloid leukemia (MDS/AML) is generally incurable and its prognosis is extremely poor. It is important to determine the predictive factors of response and survival in diseases treated with chemotherapy. Methods Twenty-nine patients who had been diagnosed of MDS/AML and had undergone chemotherapy between April 2001 and March 2008 were retrospectively analyzed. Results Of the 29 patients, 21 patients had an abnormal karyotype. Among them, 13 had complex type abnormalities and/or monosomy 7. Twenty-four patients were administered a low-dose AraC containing regimen and 5 received an AML-like regimen as the initial chemotherapy. The responses were CR4/PR2/NR23. The response rate (RR) in the patients with a normal karyotype was significantly better than in those with an abnormal karyotype (62.5% vs. 4.8%, p=0.003). Univariate analyses showed that the hemoglobin level and cytogenetic abnormalities were factors that contributed to the overall survival. Conclusion In MDS/AML, patients with a normal karyotype tended to have a better response to chemotherapy. The hemoglobin level and cytogenetic abnormalities were significant factors affecting the overall survival.

Determination of the percentage of myeloblasts in bone marrow is important for the evaluation of acute myeloblastic leukemia (AML) and related disorders. Using flow cytometry with a CD45-blast gate (FCM/CD45), 226 bone marrow aspiration samples serially collected from 71 patients with de novo AML were analyzed. Bone marrow smears were evaluated independently by pathologists who did not know the corresponding flow cytometric data in advance. Patients received remission induction followed by consolidation. The CD33+ cell percentages evaluated by FCM/CD45 were strongly correlated to the myeloblast percentages determined by microscopic examination (r=0.8360, p

The CNS has the potential to marshal strong reparative mechanisms, including activation of endogenous neurogenesis, after a brain injury such as stroke. However, the response of neural stem/progenitor cells to stroke is poorly understood. Recently, neural stem/progenitor cells have been identified in the cerebral cortex, as well as previously recognized regions such as the subventricular or subgranular zones of the hippocampus, suggesting that a contribution of cortex-derived neural stem/progenitor cells may repair ischemic lesions of the cerebral cortex. In the present study, using a highly reproducible murine model of cortical infarction, we have found nestin-positive cells in the post-stroke cerebral cortex, but not in the non-ischemic cortex. Cells obtained from the ischemic core of the post-stroke cerebral cortex formed neurosphere-like cell clusters expressing nestin; such cells had the capacity for self-renewal and differentiated into electrophysiologically functional neurons, astrocytes and myelin-producing oligodendrocytes. Nestin-positive cells from the stroke-affected cortex migrated into the peri-infarct area and differentiated into glial cells in vivo. Although we could not detect differentiation of nestin-positive cells into neurons in vivo, our current observations indicate that endogenous neural stem/progenitors with the potential to become neurons can develop within post-stroke cerebral cortex.

Semaphorins, a family of secreted and membrane-bound proteins, are known to function as repulsive axon guidance molecules. Sema4D, a class 4 transmembrane-type semaphorin, is expressed by oligodendrocytes in the central nervous system, but its role is unknown. In this study, the effects of Sema4D deficiency on oligodendrocytes were studied in intact and ischemic brains of adult mice. As observed in previous studies, Sema4D marked by beta-galactosidase in Sema4D mutant mice was localized exclusively on myelin-associated glycoprotein (MAG)-positive oligodendrocytes but not on NG2-positive oligodendrocyte progenitor cells (OPCs). Although there was no difference in the number of the latter cells between Sema4D-deficient and wild-type mice, the number of MAG-positive cells was significantly increased in the cerebral cortex of both nonischemic and postischemic brains of Sema4D-deficient mice. Cell proliferation, observed by using bromodeoxyuridine incorporation, was evident in the MAG-positive cells that developed after cerebral ischemia. These data indicate that Sema4D is involved in oligodendrogenesis during development and during recovery from ischemic injury.

Increasing evidence points to a role for circulating endothelial progenitors, including populations of CD34-positive (CD34+) cells present in peripheral blood, in vascular homeostasis and neovascularization. In this report, circulating CD34+ cells in individuals with a history of cerebral infarction were correlated with changes in neurologic function over a period of 1 year. Patients with decreased levels of CD34+ cells displayed significant worsening in neurologic function, evaluated by the Barthel Index and Clinical Dementia Rating. These results support the hypothesis that levels of circulating CD34+ cells have prognostic value for neural function, consistent with their potential role in maintaining cerebral circulation.

It has been postulated that oxygen-derived free radicals are produced in significant quantities upon reperfusion of ischemic brain and could cause brain edema and cell death. This study was undertaken in an attempt to examine the effect of recombinant human superoxide dismutase, a scavenger of superoxide radicals, on survival outcome and brain edema in gerbils undergoing 1-hour bilateral carotid occlusion and reperfusion. Superoxide dismutase was continuously infused over either 1 or 3 h of reperfusion. Neither low dose (100,000 U/kg bolus followed by 100,000 U/kg/h continuous infusion) nor high dose (100,000 U/kg bolus followed by 800,000 U/kg/h) recombinant human superoxide dismutase had an effect upon water and sodium content of whole brain at 1 h of reperfusion following 1 h of ischemia, but high-dose treatment effectively reduced brain water content at 3 h of reperfusion. All gerbils receiving high-dose treatment survived the 3 h of reperfusion, while 4 of the 7 gerbils in the control group died between 2 and 3 h of reperfusion (p less than 0.05). From this study, we conclude that prophylactic administration of superoxide dismutase can reduce the delayed vasogenic edema developing at 3 h of reperfusion and afford significant cerebroprotection in these models of transient global ischemia.

We retrospectively evaluated the efficacy of mycophenolate mofetil (MMF) in the treatment of steroid-resistant acute and chronic graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation. Thirteen patients, ten men and three women, consisted of 5 cases of acute myelogenous leukemia, 2 of acute lymphoblastic leukemia, 2 of chronic myelogenous leukemia, 2 of lymphoblastic lymphoma, and 1 case each of adult T-cell leukemia and peripheral T-cell lymphoma. The transfusions consisted of 5 peripheral blood, 7 bone marrow and 1 cord blood from 3 mothers, 4 siblings and 6 unrelated donors with conditioning treatments, including 8 total-body irradiation-based regimens, and 2 busulfan plus cyclophosphamide and 2 reduced-intensity regimens. GVHD prophylaxis included FK506 plus methotrexate (MTX) and/or antithymocyte globulin for 9 patients, and cyclosporine and MTX for 4 patients. All patients were treated with second-line MMF for steroid-refractory acute and/or chronic GVHD, and 11 patients improved. The adverse events were tolerable except for one patient in whom grade 3 neutropenia forced discontinuation of treatment. No case of non-relapse mortality occurred. We consider that MMF is beneficial and well tolerated for treatment of steroid-refractory GVHD.

The administration of CD34-positive cells after stroke has been shown to have a beneficial effect on functional recovery by accelerating angiogenesis and neurogenesis in rodent models. Granulocyte colony-stimulating factor (G-CSF) is known to mobilize CD34-positive cells from bone marrow and has displayed neuroprotective properties after transient ischemic stress. This led us to investigate the effects of G-CSF administration after stroke in mouse. We utilized permanent ligation of the M1 distal portion of the left middle cerebral artery to develop a reproducible focal cerebral ischemia model in CB-17 mice. Animals treated with G-CSF displayed cortical atrophy and impaired behavioral function compared with controls. The negative effect of G-CSF on outcome was associated with G-CSF induction of an exaggerated inflammatory response, based on infiltration of the peri-infarction area with CD11b-positive and F4/80-positive cells. Although clinical trials with G-CSF have been started for the treatment of myocardial and limb ischemia, our results indicate that caution should be exercised in applying these results to cerebral ischemia.

Frequency and clinical significance of cerebrospinal fluid (CSF) pleocytosis in hemopoietic stem cell (HSC) transplantation were surveyed. Cyclosporine (CSA)- or tacrolimus (FK506)-based regimens were used as graft-vs-host disease (GVHD) prophylaxis in allogeneic HSC transplantation. CSF pleocytosis with or without neurologic symptoms was detected in 12 of 25 patients receiving allogeneic HSC transplants but in none of 11 patients receiving autologous HSC transplants. Of the 12 patients with CSF pleocytosis, only one patient developed leukoencephalopathy later. There was a correlation between CSF cell numbers and trough levels of CSA but not with those of FK506. In patients receiving allogeneic HSC transplants, CSF pleocytosis may be relatively common and may reflect neurologic damage associated with calcineurin inhibitors.