Effect of progesterone on recovery from nerve injury during leg lengthening in rats.

We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the mRNA expression level of myelin protein P0 was analysed by quantitative real time polymerase chain reaction. On day 52 nodal length was examined by electron microscopy, followed by an examination of the compound muscle action potential (C-MAP) amplitude and the motor conduction velocity (MCV) of the tibial nerve on days 17 and 52. The P0 (a major myelin glycoprotein) mRNA expression level in the progesterone-treated group increased by 46.6% and 38.7% on days five and ten, respectively. On day 52, the nodal length in the progesterone-treated group was smaller than that in the control group, and the MCV of the progesterone-treated group had been restored to normal. Progesterone might accelerate the restoration of demyelination caused by nerve elongation by activating myelin synthesis.

Signal propagation and failure in one-dimensional FitzHugh-Nagumo equations with periodic stimuli.

We analyze the effect of additive periodic stimuli in one-dimensional FitzHugh-Nagumo equations in an excitable regime. With a suitable stimulus interval, the suppression of the pulse propagation occurs in some parameter regime. This propagation failure comes from the formation of the "death spot" where successive pulses annihilate. In the parameter regime where the solitary pulse cannot propagate in space stably, however, periodic stimuli cause a propagation of envelope of a traveling pulse under a "resonance" condition, i.e., the pulse at the leading edge disappears successively, however, an envelope is formed and propagates with keeping its shape.

Exaggerated messenger RNA expression of inflammatory cytokines in human T-cell lymphotropic virus type I-associated myelopathy.

OBJECTIVE: To investigate the expression of inflammatory cytokine messenger RNA (mRNA) in peripheral blood mononuclear cells of patients with human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM). PATIENTS: Seventeen patients with HAM, 18 HTLV-I-seropositive carriers, and 10 seronegative individuals were studied. MAIN OUTCOME MEASURE: We compared the expression of tumor necrosis factor alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha (IFN-alpha), IFN-beta, and IFN-gamma, and interleukin 1 alpha (IL-1 alpha) and IL-1 beta by reverse transcriptase-polymerase chain reaction. RESULTS: In patients with HAM, the reverse transcriptase-polymerase chain reaction products of TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha were detected in significantly higher incidences than in HTLV-I-seropositive carriers and seronegative controls. Furthermore, simultaneous mRNA expression of three or more of these four cytokines was detected in all patients with HAM compared with only 21.4% of HTLV-I-seropositive carriers. By contrast, there was no significant difference in mRNA expression of IFN-alpha, IFN-beta, and IL-1 beta among patients with HAM, HTLV-I-seropositive carriers, and HTLV-I-seronegative controls. CONCLUSIONS: An exaggerated mRNA expression of several inflammatory cytokines, including TNF-alpha, GM-CSF, IFN-gamma, and IL-1 alpha, was demonstrated in peripheral blood mononuclear cells of patients with HAM. Moreover, transcripts of these cytokines were simultaneously up-regulated in patients with HAM, suggesting that an inflammatory state in the central nervous system may be related to the pathogenesis of HAM.

Pentoxifylline down-regulates adhesion molecule expression and inflammatory cytokine production in cultured peripheral blood mononuclear cells from patients with HTLV-I-associated myelopathy.

To clarify if pentoxifylline (PTX) may have therapeutic potential for human T-cell lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), we investigated the in vitro effect of PTX on spontaneous proliferation of peripheral blood lymphocytes (SPP), as well as on the expression of adhesion molecules, such as lymphocyte function antigen-1 (LFA-1) and very late activation antigen-4 (VLA-4), and the production of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and granulocyte-monocyte colony stimulating factor (GM-CSF), in cultured PBMC from 10 HAM patients, compared with control subjects. SPP in HAM patients was significantly suppressed in a dose-dependent manner with PTX. Using flow cytometry, PTX was found to down-regulate the expression of LFA-1 and VLA-4 on CD4+ and CD8+ T cells in HAM patients as well as control subjects. However, the fall in the expression of LFA-1 and VLA-4 on CD4+ T cell population in HAM patients was higher than that of control subjects. PTX caused a significant suppression of spontaneous production of TNF-alpha by cultured PBMC of HAM patients. It also caused a small but significant suppression GM-CSF and IFN-gamma production. Collectively, our results suggest that PTX might be therapeutically effective at critical points in the immunopathogenesis of HAM.

Increased Fas-mediated cytotoxicity of CD4-positive T cells in patients with human T-lymphotropic virus type I-associated myelopathy.

Using a 51Cr release assay, we investigated Fas-mediated cytotoxicity of peripheral blood CD4+ T cells of patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) against T98G, a glioblastoma cell line which expresses Fas. Cytotoxic activity of CD4+ T cells against T98G was significantly higher in HAM patients than in controls. Moreover, when CD4+ T cells of HAM patients were preincubated with a monoclonal antibody to human Fas ligand (FasL), cytotoxic activity against T98G was significantly suppressed. These results suggest that damage to nervous tissues by the Fas/FasL system is involved in the pathogenesis of HAM.

Resistance of CD4-positive T lymphocytes to etoposide-induced apoptosis mediated by upregulation of Bcl-xL expression in patients with HTLV-I-associated myelopathy.

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) is characterized by chronic inflammation of the spinal cord. The exact mechanisms that enhance the development of chronic myelopathy remain to be determined. One such mechanism could be an altered response of peripheral blood CD4(+) T lymphocytes to apoptotic stimuli. We examined the sensitivity of these cells to apoptosis in HAM patients and control. Apoptosis was induced by etoposide, which induces mitochondria-dependent apoptosis through the release of cytochrome c from the mitochondria. The percentage of apoptotic cells that expressed hypodiploid DNA among etoposide-treated CD4(+) T lymphocytes was significantly lower in HAM patients than in the control. Western blot analysis of cell lysates derived from CD4(+) T lymphocytes demonstrated that the expression level of Bcl-xL protein was significantly higher in HAM patients than in the control. Our results indicate that peripheral blood CD4(+) T lymphocytes of HAM patients are resistant to apoptosis triggered through mitochondrial death pathway through upregulation of expression of anti-apoptotic protein, Bcl-xL. This phenomenon might contribute to the prolongation and perpetuation of the chronic inflammatory process in the spinal cord of HAM patients.

Prolactin gene expression in mouse spleen helper T cells.

Prolactin (PRL) is a single-chain polypeptide hormone that is generally secreted from prolactin cells of the anterior pituitary gland into the blood circulation. However, recent studies indicate that the gene expression of prolactin is ectopic in several tissues across several species. These studies found that lymphocytes also produce PRL, which is involved in the immunoregulatory system. Here, we searched for PRL messenger ribonucleic acid (mRNA), using the reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting in the spleens of mice at various growth stages. We also localized mouse prolactin (mPRL) and its mRNA in the spleens of 30- and 60-day-old mice by immunohistochemistry and in situ hybridization respectively. The mPRL gene was expressed in all spleen samples at 0-60 days postpartum. We localized mPRL mRNA in the sheathed artery, periarterial lymphatic sheath and the marginal zone of the spleen. Moreover, we detected mPRL in essentially the same area as its mRNA. Furthermore, we performed double-fluorescence immunohistochemical staining for mPRL and mouse CD4 that is specifically produced in helper T cells, or for mPRL and mouse CD19 or CD40 specified B cells. We colocalized mPRL immunoreactivity only in some CD4-immunopositive cells. These results clearly suggest that T cells synthesize mPRL in the mouse spleen.

Characterization of T cells transmigrating through human endothelial cells in patients with HTLV-I-associated myelopathy.

We investigated the surface markers as well as the expression of beta 2-integrin (LFA-1 beta/CD 18), in T cells migrating through human umbilical vein endothelial cells (EC) in patients with HTLV-I-associated myelopathy (HAM). No significant differences were found in the percentages of both HLA-DR+ T cells and total CD4+ cells and in the expression of beta 2-integrin between the EC-transmigrated and the EC adherent T cells. However, the percentages of HLA-DR+CD4+ cells in the transmigrated cells were significantly higher than those in the adherent cells. These results suggest that activated or HLA-DR+CD4+ T cells play an important role as the first trigger in the immunopathogenesis of HAM.

Up-regulation of iNOS mRNA expression and increased production of NO in human monoblast cell line, U937 transfected by HTLV-I tax gene.

We investigated the mRNA expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) in human T cell lymphotropic virus type I (HTLV-I) p40tax-transfected U937 cells, a human monoblast cell line. Transfection of HTLV-I p40tax U937 cells induced up-regulation of iNOS mRNA expression and subsequent NO production. Furthermore, interferon gamma (IFN-gamma) stimulation of HTLV-I p40tax-transfected U937 cells enhanced iNOS mRNA expression and NO production. The kinetics of iNOS mRNA expression and NO production indicated maximal effect at 24 and 48 hours, respectively, after culture with or without IFN-gamma. These findings suggest that HTLV-I p40tax can act as a transactivator of NO production in cells of Mo/M phi lineage. To what extent this mechanism may be involved in the pathogenesis of HTLV-I-associated diseases warrants further investigation.

Reversibility of airway obstruction in relation to prognosis in chronic obstructive pulmonary disease.

Whether reversibility in airway obstruction with beta-adrenergic stimulant is a significant determinant for the outcome was tested in 59 patients with pulmonary emphysema and chronic bronchitis. During four years of follow-up, 43 (73 percent) patients survived and 16 (27 percent) died. Initial VC. FVC, FEV1, and PaO2 were significantly smaller, and PaCO2 was significantly larger in nonsurvivors than those in survivors. After orciprenaline sulfate (10 mg in 0.5 ml solution) inhalation, VC and FEV1 increased in comparable amount between the two groups. Airway reversibility as estimated by percentage changes in FEV1 before and after the bronchodilator (reversibility index) was similar between the two groups. In the 16 nonsurvivors, hypoxemic patients had similar FEV1, FEV1/FVC, and reversibility indices as normoxemic patients. These results indicate that not airway reversibility per se but a fixed or irreversible component of airway obstruction is one of the determinants of the prognosis in pulmonary emphysema and chronic bronchitis. Chronic hypoxemia is related to neither airway obstruction nor its reversibility, while it does influence the prognosis.

Addition of cultured Schwann cells to tendon autografts and freeze-thawed muscle grafts improves peripheral nerve regeneration.

The effects of addition of Schwann cells on peripheral nerve regeneration through a novel graft material-the tendon autograft-and a conventional freeze-thawed muscle graft, were studied in the rat sciatic nerve. Adult Schwann cell cultures were established from predegenerated nerves. The Schwann cells were added to the autologous grafts by coculture (tendon autograft) or injection (freeze-thawed muscle graft). Both graft types supported adherence of the added Schwann cells. Addition of cultured Schwann cells to the two different graft models improved regeneration by increasing the rate of axonal outgrowth as compared with similar grafts without added cells.

Acute inhalation of cigarette smoke augments hypoxic chemosensitivity in humans.

Hypoxic and hypercapnic ventilatory responses were measured after two levels of acute inhalation of cigarette smoke, minimum-level nicotine smoke (smoke 1) and nicotine-containing smoke (smoke 2), in 10 normal men. Chemosensitivity to hypoxia and hypercapnia was assessed both in terms of slope factors for ventilation-alveolar PO2 curve (A) and ventilation-alveolar PCO2 line (S) and of absolute levels of minute ventilation (VE) at hypoxia or hypercapnia. Ventilatory response to hypoxia and absolute level of VE at hypoxia significantly increased from 23.5 +/- 22.6 (SD) to 38.6 +/- 31.3 l . min-1 . Torr and from 10.6 +/- 2.5 to 12.6 +/- 3.5 l . min-1, respectively, during inhalation of cigarette smoke 2 (P less than 0.05). Inhalation of cigarette smoke 2 tended to increase the ventilatory response to hypercapnia, and the absolute level of VE at hypercapnia rose from 1.42 +/- 0.75 to 1.65 +/- 0.58 l . min-1 . Torr-1 and from 23.7 +/- 4.9 to 25.5 +/- 5.9 l . min-1, respectively, but these changes did not attain significant levels. Cigarette smoke 2 inhalation induced an increase in heart rate from 64.7 +/- 5.7 to 66.4 +/- 6.3 beats . min-1 (P less than 0.05) during room air breathing, whereas resting ventilation and specific airway conductance did not change significantly. On the other hand, acute inhalation of cigarette smoke 1 changed none of these variables. These results indicate that hypoxic chemosensitivity is augmented after cigarette smoke and that nicotine is presumed to act on peripheral chemoreceptors.

Effect of brain blood flow on hypoxic ventilatory response in humans.

To assess the effect of brain blood flow on hypoxic ventilatory response, we measured arterial and internal jugular venous blood gases and ventilation simultaneously and repeatedly in eight healthy male humans in two settings: 1) progressive and subsequent sustained hypoxia, and 2) stepwise and progressive hypercapnia. Ventilatory response to progressive isocapnic hypoxia [arterial O2 partial pressure 155.9 +/- 4.0 (SE) to 46.7 +/- 1.5 Torr] was expressed as change in minute ventilation per change in arterial O2 saturation and varied from -0.16 to -1.88 [0.67 +/- 0.19 (SE)] l/min per % among subjects. In the meanwhile, jugular venous PCO2 (PjCO2) decreased significantly from 51.0 +/- 1.1 to 47.3 +/- 1.0 Torr (P less than 0.01), probably due to the increase in brain blood flow, and stayed at the same level during 15 min of sustained hypoxia. Based on the assumption that PjCO2 reflects the brain tissue PCO2, we evaluated the depressant effect of fall in PjCO2 on hypoxic ventilatory response, using a slope for ventilation-PjCO2 line which was determined in the second set of experiments. Hypoxic ventilatory response corrected with this factor was -1.31 +/- 0.33 l/min per %, indicating that this factor modulated hypoxic ventilatory response in humans. The ventilatory response to progressive isocapnic hypoxia did not correlate with this factor but significantly correlated with the withdrawal test (modified transient O2 test), which was performed on a separate day. Accordingly we conclude that an increase in brain blood flow during exposure to moderate hypoxia may substantially attenuate the ventilatory response but that it is unlikely to be the major factor of the interindividual variation of progressive isocapnic hypoxic ventilatory response in humans.

Production of granulocyte-macrophage colony stimulating factor by human T-lymphotropic virus type I-infected human glioma cells.

We investigated the production of granulocyte-macrophage colony stimulating factor (GM-CSF) by human T-lymphotropic virus type I (HTLV-I)-infected human glioma cells (KG-1-C and T98G). When glioma cells were co-cultured with HTLV-I-producing T cell lines (HCT-1 and MT-2), GM-CSF was detected in the culture supernatant. GM-CSF was produced in all the co-cultures even after several passages. In co-cultures of KG-1-C and HCT-1 cells with Millicell, the amount of GM-CSF produced in the supernatant was almost as low as in the culture of HCT-1 alone. Moreover, for co-cultures of KG-1-C and HCT-1 or MT-2 cells, the production of GM-CSF was significantly suppressed in the presence of IgG from patients with HAM. Double-label immunostaining showed that GM-CSF-producing glioma cells always were stained by a monoclonal antibody against HTLV-I p19, indicating that HTLV-I infection of glioma cells caused GM-CSF production. These data suggest that human glial cells infected with HTLV-I gain the ability to produce cytokines.

Successful application of pentoxifylline in the treatment of HTLV-I associated myelopathy.

Fifteen patients with human T-cell lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial by oral administration of pentoxifylline (PTX). Motor function, neurological evaluation, immunological markers and parameters were evaluated after four weeks. In 13 of the 15 patients, motor disability, especially spasticity, improved substantially. PTX suppressed spontaneous proliferation of peripheral blood mononuclear cells in 14 of the 15 patients at four weeks. No adverse effect was observed. We concluded that PTX may be a safe and beneficial agent for the treatment of HAM.

Characterization of adherent T cells to human endothelial cells in patients with HTLV-I-associated myelopathy.

To clarify the phenomenon of increased adherence of T cells to endothelial cells (EC) in patients with HTLV-I-associated myelopathy (HAM), we determined the surface markers and expression of lymphocyte function antigen-1 (LFA-1) in T cells adherent or nonadherent to EC. The percentage of activated or HLA-DR+ T cells and the expression of LFA-1 in the adherent cell population were significantly higher than those in the nonadherent cell population. Moreover, the CD4 to CD8 ratio of the HLA-DR+ cells in the EC-adherent T cells was significantly higher than that in the nonadherent cells. Collectively, these results indicate that increased adherence of T cells to EC in HAM patients is based on the increase of activated T cells with high density LFA-1 expression in the peripheral blood. Moreover, CD4+ HLA-DR+ cells exhibited more adhesive activity to EC than CD8+ HLA-DR+ cells, suggesting that activated CD4+ cells, rather than activated CD8+ cells, may be important as the first trigger for T cell-infiltration to the central nervous system in the immunopathogenesis of HAM.

Heparin treatment in patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy: a preliminary study.

Ten patients with HTLV-I-associated myelopathy (HAM) were treated in an uncontrolled preliminary trial of heparin. In 7 patients, motor dysfunction improved substantially and the effect continued for more than a month after the discontinuation of therapy. Sensory and urinary disturbances also improved in 3 of 4 and in 2 of 10 patients, respectively. Heparin did not alter the subsets of peripheral blood lymphocytes nor the titers of anti-HTLV-I antibodies in serum and cerebrospinal fluid. Spontaneous proliferation of peripheral blood lymphocytes, however, was depressed significantly (P < 0.05) in all cases. Heparin therapy has some advantages in cost, ease of administration and fewer side effects compared to other therapies such as plasmapheresis and interferon-alpha. We conclude that heparin can be administered safely to HAM, and that a double-blind placebo-controlled trial is warranted to determine its efficacy in HAM.

Antiproliferative factor against the human glioblastoma cell line T98G identified in culture supernatants of CD4+ cells from patients with HTLV-I-associated myelopathy.

We investigated culture supernatants of peripheral blood mononuclear cells (MNC) derived from patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) for antiproliferative activity against the human glioblastoma cell line T98G. When T98G cells were cultured with condition medium containing culture supernatants of MNC from patients with HAM, the proliferation of T98G cells was significantly suppressed, compared with that of supernatants from HTLV-I seropositive carriers or seronegative controls. To clarify which population of MNC produced the antiproliferative humoral factor for T98G cells, we separated MNC into macrophage-depleted or B cell depleted populations, and further to both CD4+ and CD8+ T cells by using the panning method or plastic adherence. These studies demonstrated that the antiproliferative activity was mediated by a humoral factor produced by T cells, specifically CD4+ cells. This activity was blocked by a neutralizing monoclonal antibody against interferon-gamma (IFN-gamma). Moreover, IFN-gamma levels were elevated in the culture supernatants of CD4+ cells from HAM patients. Thus, the antiproliferative activity against T98G cells is mainly due to IFN-gamma derived from CD4+ cells of patients with HAM.

Elevated serum levels of soluble E- and L-selectin in patients with human T-cell lymphotropic virus type I-associated myelopathy.

We compared soluble E-selectin (sE-selectin) and L-selectin (sL- selectin) levels in sera and cerebrospinal fluid (CSF) of 30 patients with human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM), with those of 10 patients with the relapsing-remitting form of multiple sclerosis (MS), and 16 patients with other neurological diseases (OND). Serum levels of both sE-selectin and sL-selectin, as measured by enzyme-linked immunosorbent assay, were significantly elevated in patients with HAM, compared to patients with OND. In addition, serum levels of sL-selectin were significantly elevated in HAM patients compared to MS patients. No significant difference was found in CSF levels of sL-selectin between HAM patients and controls. However, HAM patients who had received blood transfusions had significantly higher CSF levels of sL-selectin than HAM patients without a past history of transfusions, suggesting that HAM patients with past history of transfusion have a more active immunological state in the central nervous system. sE-selectin was not detected in CSF of HAM patients and controls. This finding might be based on exaggerated inflammatory conditions following increased attachment of lymphocytes to activated endothelial cells in HAM patients.

Importance of immune deviation toward Th1 in the early immunopathogenesis of human T-lymphotropic virus type I-associated myelopathy.

Although the principal neuropathological feature of human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) is chronic inflammation of the spinal cord, characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration, the precise mechanisms by which HTLV-I infection causes chronic inflammation of the spinal cord are still obscure. In patients with HAM, peripheral blood CD4(+)T lymphocytes, particularly HTLV-I-infected CD4(+)T lymphocytes, have increased adherent activity to endothelial cells and transmigrating activity through basement membranes. In addition, the profile of cytokine expression suggests increased numbers of Th1 cells in peripheral blood CD4(+)T lymphocytes of patients with HAM. These findings strongly suggest that immune deviation toward Th1, which might be based on high viral load of HTLV-I, plays an important role in tissue damage in the central nervous system of patients with HAM. We herein emphasize the importance of activated Th1 cells as the first trigger in the immunopathogenesis of HAM.