A case of improvement of clozapine-induced low leukocyte counts by adenine, cepharanthin and ninjin-yoei-to in a patient with treatment-resistant schizophrenia.

BACKGROUND: Although clozapine is the optimal drug for patients with treatment-resistant schizophrenia, the drug has harmful adverse effects such as leukopenia. Adenine and cepharanthine are known to be effective for radiation- or drug-induced leukopenia. Furthermore, ninjin-yoei-to, a Chinese herbal medicine, augments the production of granulocyte-macrophage colony-stimulating factor. Thus, these drugs may be useful for clozapine-induced leukopenia. CASE PRESENTATION: A 21 years-old woman with schizophrenia was hospitalized for initiation of clozapine treatment. Despite concomitant use of adenine, cepharanthine, and lithium carbonate having activities of increasing leukocytes, a decrease in leukocyte counts occurred after the initiation of clozapine. Additional administration of ninjin-yoei-to increased leukocyte counts, which prevented the development of leukopenia. CONCLUSIONS: This is the first case that concomitant use of adenine, cepharanthin, and ninjin-yoei-to exhibited the effectiveness of reversing the decrease in leukocytes caused by clozapine. Monitoring leukocyte counts and preventing leukopenia are essential for successful treatment with clozapine for refractory schizophrenia. These medicines may be a potential option for preventing clozapine-induced leukopenia.

Prophylactic Treatment with Intravenous Immunoglobulin Attenuates Experimental Optic Neuritis in Mice.

Optic neuritis is characterized by optic nerve inflammation, demyelination and axonal loss. Intravenous immunoglobulin (IVIg) has been reported to be effective for steroid-resistant patients. However, there is no report investigating the histopathological efficacy of IVIg in optic neuritis models. In this study, we examined the effects of IVIg on optic neuritis of experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune optic neuritis (EAON). Inflammation, demyelination and axonal loss were assessed in the optic nerve sections. IVIg showed dose-dependent prevention of clinical symptoms in EAON. IVIg provided an anti-inflammatory effect in both EAE and EAON, associated with improved demyelination. Axonal loss in EAE was also significantly attenuated. These results suggest that IVIg has neuroprotective properties in experimental optic neuritis, and is a promising new treatment for optic neuritis.

Immunoglobulin G Enhances Generation of Inducible T Regulatory Cells and Increases Their Regulatory Function.

Intravenous immunoglobulin (IVIg) has been shown to be effective in the treatment of a variety of autoimmune diseases. To clarify the role of T regulatory cells (Tregs) in the immunoregulatory effect of IVIg, we focused on human inducible T regulatory cells (iTregs) and investigated the mechanism of action of IVIg. When immunoglobulin G (IgG) was added to a culture system that differentiates iTregs from anti-CD3 antibody activated CD4+CD25- T cells in the presence of syngeneic immature dendritic cells, interleukin (IL)-2 and transforming growth factor-ß (TGF-ß), the expression of forkhead box P3 (FoxP3), which is the master transcription factor for Tregs in CD4+CD25+ T cells, increased in an IgG concentration-dependent manner. The expression of FoxP3 in iTregs in the 20 mg/mL IgG group was twice as high as that in the saline group. iTregs that highly expressed FoxP3 not only partially suppressed the polyclonal proliferative response of T cells derived from the same individual but also produced significantly more inhibitory cytokines IL-10 and TGF-ß. The ability of IgG to enhance iTregs differentiation was also observed in the Fc fragment, but not in the F(ab')2 fragment. These results suggest the clinical regulation of immune responses by IVIg administration may contribute at least to enhancing the differentiation of iTregs and partial immunosuppressive functions.

Anti-inflammatory effects of activated protein C on human dendritic cells.

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti-apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF-α and IL-6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL-12 and anti-inflammatory cytokine IL-10. Interestingly, treatment with 5 µg/mL, but not 25 µg/mL, of APC significantly enhanced production of IL-10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co-stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti-inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF-α and/or IL-6 production.

Potent cough suppression by physiologically active substance in human plasma.

Human plasma contains wide variety of bioactive proteins that have proved essential in therapeutic discovery. However many human plasma proteins remain orphans with unknown biological functions. Evidences suggest that some plasma components target the respiratory system. In the present study we adapted heparin affinity chromatography to fractionate human plasma for functional bioassay. Fractions from pooled human plasma yielded particular plasma fractions with strong cough suppressing effects. Purification yielded a fraction that was finally identified as an activated blood coagulation factor fXIa using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). The fraction almost completely suppressed coughs induced by either chemical or mechanical stimulation applied to larynx or bifurcation of guinea-pig trachea. Cough suppressing effect of the fraction and commercially available fXIa were one million times stronger than codeine and codeine only partially suppressed the mechanically triggered coughing in animal model. Recent reviews highlighted prominent shortcomings of current available antitussives, including narcotic opioids such as codeine and their unpleasant or intolerable side effects. Therefore, safer and more effective cough suppressants would be welcome, and present findings indicate that fXIa in human plasma as a very promising, new therapeutic candidate for effective antitussive action.

[Enhancement of regulatory T cell induction by intravenous S-sulfonated Immunoglobulin during the treatment of experimental autoimmune encephalomyelitis].

Intravenous immunoglobulin (IVIg) has been shown to be effective for a variety of autoimmune diseases. Despite its widespread use and therapeutic success, the precise mechanisms for the anti-inflammatory therapeutic effects of IVIg are not well understood. In particular, few reports have examined the mechanism of IVIg on regulatory T cells (Treg: CD4(+)CD25(+)FoxP3(+) T cells). In the present study, to clarify the effect of intravenous S-sulfonated immunoglobulin (S-IVIg) on Treg, we investigated experimental autoimmune encephalomyelitis (EAE), the representative animal model of autoimmune disease. First, when we evaluated the effect of S-IVIg in an acute EAE model, the prophylactic treatment of S-IVIg dose-dependently controlled the symptoms of EAE. Next, we measured Treg in EAE mice spleen by flow cytometry. The percentage of Treg in S-IVIg-treated mice was significantly increased compared with Saline-treated mice. Finally, in reinduced EAE, S-IVIg not only prevented EAE progression, but also increased the percentage of Treg in the spleen. The increase in percentage of Treg in S-IVIg-treated EAE might be associated with protection against EAE. These observations provide important evidence that IVIg is effective in T-cell-mediated control of autoimmunity.

Intravenous immunoglobulin therapy prevents development of autoimmune encephalomyelitis and suppresses activation of matrix metalloproteinases.

Although intravenous immunoglobulin (IVIG) has been reported to improve the status of expanded disability status scale (EDSS) of multiple sclerosis (MS) patients and reduce the annual relapse rate, some studies did not find its beneficial effects. In the present study, using an animal model for MS, we found that prophylactic, but not therapeutic, treatment successfully suppressed the disease development. During the search for factors involved in the disease suppression by IVIG, we obtained evidence suggesting that IVIG exerts its function, at least in part, by suppressing activation of matrix metalloproteinases (MMP)-2 and -9. Gelatin zymography revealed that gelatinase activities were suppressed by IVIG treatment in the spinal cord, but not in plasma. This finding raises the possibility that IVIG blocks MMP activities at the interface between the blood stream and CNS. With in situ zymography, we also observed that gelatinase activities were expressed mainly in astrocytes in the inflamed spinal cord of control rats and that this expression was attenuated by the treatment. These findings provide useful information to set optimal conditions for IVIG treatment of MS and to obtain more beneficial effects.

Effect of biodegradable fibrin scaffold on survival, migration, and differentiation of transplanted bone marrow stromal cells after cortical injury in rats.

OBJECT: In this study the authors' aim was to assess whether fibrin matrix could act as an injectable, valuable scaffold in bone marrow stromal cell (BMSC) transplantation for injured CNS tissue. METHODS: Both clotting time and 3D structure of fibrin matrix were analyzed with various concentrations of fibrinogen and CaCl(2). The BMSCs were harvested from green fluorescent protein-transgenic mice and cultured. A cortical lesion was produced in rats by application of a very cold rod to the right cerebral hemisphere. The BMSCs, fibrin matrix, or BMSC-fibrin matrix complex was transplanted into the lesion though a small bur hole 7 days after the insult. Using immunohistochemical analysis, the authors evaluated the survival, migration, and differentiation of the transplanted cells 4 weeks after transplantation. RESULTS: Based on in vitro observations, the concentrations of fibrinogen and CaCl(2) were fixed at 2.5 mg/ml and 2 microM in animal experiments, respectively. Fibrin matrix almost completely disappeared 4 weeks after transplantation. However, immunohistochemical analysis revealed that fibrin matrix exclusively enhanced the retention of the transplanted cells within the lesion, migration toward the lesion boundary zone, and differentiation into the neurons and perivascular cells. CONCLUSIONS: Injectable fibrin matrix enhanced the survival, migration, and differentiation of the BMSCs transplanted into the cortical lesion in rats. The authors believe that it is one of the promising candidates for a potential, minimally invasive scaffold for CNS disorders. The present findings strongly suggest that such a strategy of tissue engineering could be a therapeutic option for CNS regeneration in patients with CNS injuries.

Fibrin matrix provides a suitable scaffold for bone marrow stromal cells transplanted into injured spinal cord: a novel material for CNS tissue engineering.

Recent basic experiments have strongly suggested that cell transplantation therapy may promote functional recovery in patients with spinal cord injury (SCI). However, a safe and efficient transplantation technique still remains undetermined. This study, therefore, was aimed to clarify whether fibrin matrix could be a useful scaffold in bone marrow stromal cell (BMSC) transplantation for the injured spinal cord. To clarify the issue, three-dimensional structure of fibrin matrix was assessed and the green fluorescent protein (GFP)-expressing BMSC were cultured in fibrin matrix. The rats were subjected to spinal cord hemisection at T8 level, and the vehicle, BMSC or BMSC-fibrin matrix construct was implanted into the cavity. Neurologic function was serially evaluated. Using immunohistochemistry, we evaluated the survival, migration and differentiation of the transplanted cells at 4 weeks after transplantation. In the initial in vitro study, the BMSC could survive in fibrin matrix for 2 weeks. The animals treated with the BMSC-fibrin matrix construct showed significantly more pronounced recovery of neurologic function than vehicle- or BMSC-treated animals. Fibrin scaffold markedly improved the survival and migration of the transplanted cells. There was no significant difference in the percentage of cells doubly positive for GFP and microtubule-associated protein 2 between the animals treated with BMSC-fibrin matrix construct and those treated with BMSC, but a certain subpopulation of GFP-positive cells morphologically simulated the neurons in the animals treated with BMSC-fibrin matrix construct. These findings strongly suggest that fibrin matrix may be one of the promising candidates for a potential, minimally invasive scaffold for injured spinal cord, and that such strategy of tissue engineering could be a hopeful option in regeneration therapy for patients with SCI.

Tissue factor pathway inhibitor is highly susceptible to chymase-mediated proteolysis.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that primarily inhibits the extrinsic pathway of blood coagulation. It is synthesized by various cells and its expression level increases in inflammatory environments. Mast cells and neutrophils accumulate at sites of inflammation and vascular disease where they release proteinases as well as chemical mediators of these conditions. In this study, the interactions between TFPI and serine proteinases secreted from human mast cells and neutrophils were examined. TFPI inactivated human lung tryptase, and its inhibitory activity was stronger than that of antithrombin. In contrast, mast cell chymase rapidly cleaved TFPI even at an enzyme to substrate molar ratio of 1:500, resulting in markedly decreased TFPI anticoagulant and anti-(factor Xa) activities. N-terminal amino-acid sequencing and MS analyses of the proteolytic fragments revealed that chymase preferentially cleaved TFPI at Tyr159-Gly160, Phe181-Glu182, Leu89-Gln90, and Tyr268-Glu269, in that order, resulting in the separation of the three individual Kunitz domains. Neutrophil-derived proteinase 3 also cleaved TFPI, but the reaction was much slower than the chymase reaction. In contrast, alpha-chymotrypsin, which shows similar substrate specificities to those of chymase, resulted in a markedly lower level of TFPI degradation. These data indicate that TFPI is a novel and highly susceptible substrate of chymase. We propose that chymase-mediated proteolysis of TFPI may induce a thrombosis-prone state at inflammatory sites.

Laminin-induced autoimmune myositis in rats.

The present study aimed to examine if immunization with laminin causes myositis in rats and whether the pathologic findings mirror human polymyositis and dermatomyositis. Rats were immunized with an emulsion of laminin and complete Freund's adjuvant. As a result, muscle fiber necrosis with infiltrating macrophages was frequently observed and mononuclear cells were observed in the endomysium. These mononuclear cells were composed of CD4+ cells, CD8+ T cells, and macrophages. CD4+ cells and CD8+ T cells were mainly located in the endomysium, whereas a large number of macrophages were located in the endomysium and infiltrating muscle fibers. A small number of B cells, detected by immunohistochemical staining, were mainly located in the perimysium. The nonnecrotic muscle fiber to which CD4+ T cells, CD8+ T cells, and perforin+ cells adhered was negative for antimerosin and antidystrophin antibodies. Muscle fiber necrosis in rats immunized with laminin may occur after denaturation of basement membrane proteins. In conclusion, the immunization with laminin induces moderate to severe myositis. We suggest that laminin may be an important antigen for connective tissue diseases such as polymyositis and dermatomyositis.

The effect of human tissue factor pathway inhibitor-2 on the growth and metastasis of fibrosarcoma tumors in athymic mice.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits the plasmin- and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion, and metastasis. To directly assess its role in tumor growth and metastasis in vivo, we stably transfected HT-1080 fibrosarcoma cells expressing either fully active wild-type human TFPI-2 (WT) or inactive R24Q TFPI-2 (QT) and examined their ability to form tumors and metastasize in athymic mice in comparison to mock-transfected cells (MT). MT and QT fibrosarcoma tumors grew 2 to 3 times larger than WT tumors. Tumor metastasis was confined to the lung and was observed in 75% of mice treated with either MT or QT cells, whereas only 42% of mice treated with WT cells developed lung metastases. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of each tumor group revealed 3- to 6-fold lower levels of murine vascular endothelial growth factor gene expression in WT tumors in relation to either MT or QT tumors. Comparative tumor gene expression analysis revealed that several human genes implicated in oncogenesis, invasion, metastasis, apoptosis, and angiogenesis had significantly altered levels of expression in WT tumors. Our collective data demonstrate that secretion of inhibitory TFPI-2 by a highly metastatic tumor cell markedly inhibits its growth and metastasis in vivo by regulating pericellular extracellular matrix (ECM) remodeling and angiogenesis.