Phosphorylation of Smad2/3 at the specific linker threonine residue indicates slow-cycling esophageal stem-like cells before re-entry to the cell cycle.

UNLABELLED: The stem cell compartment in the esophageal epithelium is possibly located in the basal layer. We have identified significant expression of Smad2/3, phosphorylated at specific linker threonine residues (pSmad2/3L-Thr), in the epithelial cells of murine stomach and intestine, and have suggested that these cells are epithelial stem cells. In this study, we explore whether pSmad2/3L-Thr could serve as a biomarker for esophageal stem cells. We examined esophageal tissues from normal C57BL/6 mice and those with esophagitis. Double immunofluorescent staining of pSmad2/3L-Thr with Ki67, CDK4, p63, or CK14 was performed. After immunofluorescent staining, we stained the same sections with hematoxylin-eosin and observed these cells under a light microscope. We used the 5-bromo-2-deoxyuridine (BrdU) labeling assay to examine label retention of pSmad2/3L-Thr immunostaining-positive cells. We collected specimens 5, 10, 15 and 20 days after repeated BrdU administrations and observed double immunofluorescent staining of pSmad2/3L-Thr with BrdU. In the esophagus, pSmad2/3L-Thr immunostaining-positive cells were detected in the basal layer. These cells were detected between Ki67 immunostaining-positive cells, but they were not co-localized with Ki67. pSmad2/3L-Thr immunostaining-positive cells showed co-localization with CDK4, p63, and CK14. Under a light microscope, pSmad2/3L-Thr immunostaining-positive cells indicated undifferentiated morphological features. Until 20 days follow-up period, pSmad2/3L-Thr immunostaining-positive cells were co-localized with BrdU. pSmad2/3L-Thr immunostaining-positive cells significantly increased in the regeneration phase of esophagitis mucosae, as compared with control mice (esophagitis vs. CONTROL: 6.889 ± 0.676/cm vs. 4.293 ± 0.659/cm; P < 0.001). We have identified significant expression of pSmad2/3L-Thr in the specific epithelial cells of murine esophagi. We suggest that these cells are slow-cycling epithelial stem-like cells before re-entry to the cell cycle.

Concentration of clarithromycin and 14-R-hydroxy-clarithromycin in plasma of patients with Mycobacterium avium complex infection, before and after the addition of rifampicin.

Clarithromycin (CAM) and rifampicin (RFP) have both been recognized to be effective antibiotic agents against Mycobacterium avium complex (MAC) infection. Rifamycin derivatives including RFP and rifabutin modulate the CAM metabolism by inducing the hepatic cytochrome p-450 3A4. To clarify the effect of RFP on the CAM metabolism, we measured the plasma concentration of CAM and 14-R-hydroxyclarithromycin (M-5), the major metabolite of CAM, in 9 patients suffering from MAC infection before and after the addition of RFP. After the addition of RFP, the mean plasma concentration of CAM significantly decreased, while that of M-5 did not. In addition, the amount of CAM + M-5 concentration also significantly decreased. As M-5 is less effective against MAC infection than CAM, more attention should thus be paid to the plasma CAM concentration in patients administered CAM and RFP concomitantly.

Diaphragm pacing with the spinal cord stimulator.

BACKGROUND: Diaphragm pacing with electrical stimulation of the phrenic nerve is an established treatment for central hypoventilation syndrome. The device, however, is not readily available, at least, in Japan. We applied the spinal cord stimulator for pain control to phrenic nerve stimulation. The purpose of this study is to evaluate the efficacy and feasibility of phrenic pacing with the compromise method. METHOD AND PATIENTS: We implanted a spinal cord stimulator in five patients with chronic central hypoventilation. The stimulation electrode was placed along the phrenic nerve in the neck, and the device was implanted in the anterior chest. We used the cyclic mode, and set the parameters at 1 second ramp up, 2 seconds on, 3 seconds off. The pulse width and the frequency were set at 150 microsec and 21 Hz respectively. The amplitude was adjusted to obtain sufficient tidal volume and to maintain PaCO2 at around 40 mmHg. FINDINGS: During the follow-up period from 3 to 34 months (mean 23), stable and sufficient ventilation were observed in all patients without complications. INTERPRETATION: Although longer follow-up is necessary, diaphragm pacing with the spinal cord stimulator is feasible for treatment of central hypoventilation syndrome.

[Pulmonary Mycobacterium Kansasii infection in the southern area of Fukuoka prefecture].

Cases of pulmonary infection caused by Mycobacterium kansasii (Mk) in our hospital located at the mid-northern area of the Kyushu district, which is in the southern part of Fukuoka prefecture were evaluated. Mk infection is not so rare in other areas of Japan, such as Tokyo and Kinki district, however, there has been no published report on the disease from the Kyusyu district. Therefore, the frequency and the clinical features of our cases of Mk infection were analyzed. During 17 years from 1982 to 1998, there were 14 patients of Mk infection out of 241 nontuberculous mycobacteriosis (NTM). There were 595 patients of culture-positive pulmonary tuberculosis without prior treatment (Tbc). The proportion of Mk/Tbc was 2.4% and that of Mk/NTM was 5.8%. During the period A (from 1982 to 1994) the ratio of Mk/Tbc was 5/462 (1.1%), while on the other side that of Mk/Tbc during the period B (from 1995 to 1998), it was 9/133 (6.8%), which was significantly (P < 0.01) higher compared with that in the period A. Although the patients of Mk infection in our hospital had been rare until 1994, from the results mentioned above, it was considered that the frequency of Mk infection in our hospital has increased to some extent since 1995. One of the characteristics in our cases was that the ratio of female (42.9%) was relatively high. All the female patients were considered to be compromised hosts. The results of the drug resistance tests were consistent with the other reports in our country. By the combination treatment including rifampicin as the major drug, the negative conversion of culture were obtained within 2 months in all our cases.

[Left Sylvian fissure meningioma in a one-year-eight-month old child].

We report a case of a Sylvian fissure meningioma in a one-year-eight-month old child who experienced the onset of a convulsive seizure. He had no neurological deficit and no developmental disorders. Computed tomography (CT) and magnetic resonance imaging (MRI) showed a large left temporal tumor which was well enhanced and without dural attachment. Angiography revealed a slight tumor stain in the left Sylvian fissure supplied by branches of the internal carotid artery. Total removal of the tumor was performed, and we found that the tumor had no dural attachment, but was strongly attached to the M2 segment of the left middle cerebral artery. Pathological examinations revealed it to be a fibrous meningioma without malignancy. This is the youngest case among the reported five pediatric deep Sylvian meningiomas. Introducing this case, we discuss the clinical features of pediatric meningiomas.

[Selective peripheral denervation for spasmodic torticollis involving the levator scapulae muscle].

Patients with laterocollis or rotatory type torticollis tend to show abnormal contraction of the levator scapulae muscle and the scalene muscles. These muscles are innervated from the anterior branches of the cervical spinal nerves. Because of this, the traditional Bertrand operation dealing with posterior branches does not adequately affect the symptoms of laterocollis. The authors report selective denervation of the levator scapulae muscle in three patients and discuss its rationale. All the three patients underwent denervation of both the C1-C6 posterior spinal rami and the branches from the C3 and C4 anterior rami to the levator scapulae muscle. We added myotomy of the scalene muscle in one patient, and denervation of the omohyoid muscle which is innervated from the ansa cervicalis and the descending branch of the hypoglossal nerve. The pre/post-operative Tsui scores were 12/4, 15/1, and 14/3 respectively. There were no complications. We conclude that selective peripheral denervation of the levator scapulae muscle is safe and effective in the treatment of laterocollic type torticollis.

Recurrent chest opacities in a patient with thromboembolic pulmonary hypertension.

We describe a 42-year-old man with chronic thromboembolic pulmonary hypertension associated with protein S deficiency. He presented with unusual roentgenographic findings of migratory pulmonary infiltrates.

Release of nitric oxide and expression of constitutive nitric oxide synthase of human endothelial cells: enhancement by a 14-membered ring macrolide.

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca2+]i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca2+]i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.

Simultaneous measurement of the phase and group indices and the thickness of transparent plates by low-coherence interferometry.

We propose and demonstrate a novel technique for simultaneous measurement of the phase index, n(p) , the group index, n(g) , and the thickness, t , of transparent plates by use of a low-coherence interferometer. The output light from a superluminescent diode is focused upon the front plane of a transparent plate that is used as the sample. The sample stage is subsequently moved until the light is focused upon the rear plane of the plate. Measurement of the stage movement distance and the corresponding optical path difference allows us to determine both n(p) and n(g) . By placing the sample between two glass plates, we measured n(p) , n(g) , and t simultaneously, with an error of 0.3% or less, for nearly 1-mm-thick transparent plates, including glass and electro-optic crystals.

The role of nitric oxide in human pulmonary artery endothelial cell injury mediated by neutrophils.

Human endothelial cells are injured by the action of leukocytes. We investigated the role of nitric oxide (NO) in the induction of injury to human pulmonary artery endothelial cells. NO has been a putative source of cytotoxic reactive oxygen species in some settings. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. This effect was augmented by treatment with carboxy-PTIO, which traps NO in the medium, or with L-NAME, an inhibitor of NO synthase. When endothelial cells were incubated with neutrophils stimulated by phorbol myristate acetate, an activator of protein kinase C, endothelial cell damage was further enhanced and the amount of NO in the medium was decreased. Dibutyryl cyclic AMP, a cell-permeable analogue of cyclic AMP, protected against neutrophil-induced endothelial cell injury and increased NO release into the medium. The effects of dibutyryl cyclic AMP were abrogated by treatment with H-89, a potent inhibitor of cyclic AMP-dependent protein kinase. The protective effect on neutrophil-induced endothelial cell injury by dibutyryl cyclic AMP was abolished by addition of carboxy-PTIO or L-NAME. Thus, our studies suggest that NO, presumably released from endothelial cells, protects against endothelial injury by activated neutrophils and the protective effect by cyclic AMP during coculture with activated neutrophils is mediated through the action of NO. However, when monocytes activated by lipopolysaccharide and IFN-gamma were used instead of neutrophils, endothelial cells were likewise injured, but a much higher level of NO was detected and injury was diminished by addition of carboxy-PTIO to the medium. These observations suggest that the high levels of NO released by activated monocytes contribute to endothelial injury, whereas low levels of NO protect endothelial cells against injury by neutrophils.

Neutrophil-induced endothelial cell damage: inhibition by a 14-membered ring macrolide through the action of nitric oxide.

Macrolide antibiotics have been used worldwide for about 40 years. The clinical effectiveness of oral erythromycin for diffuse panbronchiolitis has been established and erythromycin seems to act not only as an antibacterial but also as an anti-inflammatory agent. We investigated the effect of 14-membered ring macrolides, erythromycin and clarithromycin, on human neutrophil functions and endothelial cell damage induced by neutrophils. The superoxide production of neutrophils and Ca2+ influx into neutrophils induced by N-formyl-methionyl-leucyl-phenylalanine was inhibited by treatment with erythromycin but not by treatment with clarithromycin. When endothelial cells were cocultured with neutrophils, nitric oxide (NO) presumably released from endothelial cells were enhanced by treatment with erythromycin but not by treatment with clarithromycin and endothelial cell injury induced by neutrophils was ameliorated by addition of erythromycin but not by clarithromycin. The reduction of neutrophil-induced endothelial cell injury by erythromycin was abolished by treatment with carboxy-PTIO which traps NO in the medium. Moreover, nitrite in the medium in which endothelial cells were incubated with neutrophils was enhanced by treatment with erythromycin and the enhancement of nitrite by erythromycin was partially cancelled by addition of H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). Erythromycin seems to ameliorate neutrophil-induced endothelial cell injury by affecting not only neutrophil functions but the release of NO from endothelial cells through the action of PKA. The usefulness for the treatment of diseases worsened by the interaction between neutrophils and endothelium might be different among 14-membered ring macrolides.

The role of neutrophil elastase in human pulmonary artery endothelial cell injury.

Neutrophils are thought to play a key role in tissue injury. We investigated the role of human neutrophil-derived elastase in the induction of injury to human pulmonary artery endothelial cells. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity, thrombomodulin, and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. Attachment alone of neutrophils to endothelial cells appeared to induce activation because elastase release and N-formyl-mentionyl-leucyl-phenylalanine (fMLP)-induced superoxide (O2) production from neutrophils incubated with endothelial cells were greater than from neutrophils only. When endothelial cell were incubated with neutrophils stimulated by fMLP or phorbol myristate acetate, the amount of elastase in the medium and endothelial cell damage was further enhanced. However, when neutrophils were blocked from direct attachment to endothelial cells using a membrane filter, endothelial cell damage was ameliorated, while exogenous neutrophil elastase and medium containing neutrophil-released elastase did not induce endothelial cell injury. An inhibitor of neutrophil elastase, ONO-5046 Na, as well as erythromycin, which reduces neutrophil-derived elastase, dramatically inhibited neutrophil-induced endothelial cell injury. Superoxide dismutase (SOD) partially inhibited injury. Injury was completely inhibited by treatment with a combination of ONO-5046 Na and SOD. These results suggest that attachment of neutrophils to endothelial cells is important for endothelial cell damage and that neutrophil-derived elastase plays an important role in endothelial cell injury in combination with O2. In addition, ONO-5046 Na and erythromycin may be useful in treating diseases worsened by excessive neutrophil activity.

[Clinical efficacy of imipenem/cilastatin sodium for respiratory infections in patients with lung cancer].

Imipenem/cilastatin sodium (IPM/CS) was administered to 102 patients with respiratory tract infections and lung cancer. Patients with other serious diseases were excluded and a total of 73 patients were enrolled. They were divided into 12 patients who underwent surgery (operated group) and 61 who did not (non-operated group); the latter group included 28 patients treated with anticancer agents or radiation therapy (treated group) and 33 untreated patients (untreated group). IPM/CS was effective in 75% of the patients, both with and without surgery. The drug was effective in 81% of the treated group, although many of the patients had Stage III or more advanced cancer, as well as bronchial occlusion. IPM/CS was also effective in 69% of the untreated group, although many of the patients have serious infections and a PS (Performance Status) of 3 or greater. Thus, IPM/CS treatment achieved good results. Bacteriological studies showed that 3 out of 4 strains in the operated group and 16 out of 18 in the non-operated group were eliminated. Safety was evaluated in all patients. Two patients (2%) experienced side effects and two others (2%) showed abnormal clinical findings, but the symptoms were mild and resolved after discontinuation or completion of therapy. In conclusion, IPM/CS was very effective for treating respiratory infections in patients with lung cancer.

Inhibition by erythromycin of human pulmonary artery endothelial cell injury induced by human neutrophils.

Neutrophils are thought to play a key role in tissue injury. We investigated the role of human neutrophils in the induction of injury to the human pulmonary artery endothelial cells and the effect of erythromycin on neutrophil-induced endothelial cell damage. Incubation of unstimulated neutrophils with endothelial cells increased the release of lactate dehydrogenase (LDH) activity and preloaded fura-2 from endothelial cells. When neutrophils were activated by phorbol myristate acetate, the release of LDH and fura-2 was enhanced further. Superoxide dismutase partially inhibited the release of LDH and fura-2 induced by neutrophils, whereas erythromycin markedly inhibited the release of endothelial cell LDH and fura-2 induced by neutrophils. These results suggest that endothelial cell injury is, at least in part, mediated by the action of superoxide and that erythromycin protects against neutrophil-induced endothelial cell injury.

Lung cancer cell lines inhibit leukotriene B4 production by human polymorphonuclear leukocytes at the level of phospholipase A2.

We studied cellular interactions between human polymorphonuclear leukocytes (PMN) and lung cancer cell lines by investigating the influence of cancer cells on the production of leukotriene B4 (LTB4) and superoxide anion (O2-) by stimulated PMN. Of the nine cancer cell lines established from human lung cancers that we examined, H23 cells showed the highest LTA4 hydrolase activity. When PMN were stimulated by the calcium ionophore A23187 in the presence of H23 cells, the production of LTB4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), and 12(S)-hydroxyeicosatetraenoic acid (12-HETE) decreased in a dose-dependent manner. On the contrary, H23 did not inhibit O2- production by PMN. Two other cell lines (N417 and Q9) caused similar inhibition of LTB4 production by PMN. These three cancer cell lines alone did not generate any metabolites of the arachidonic acid (AA) lipoxygenase pathway or any O2- upon stimulation with A23187 alone. The addition of AA dose-dependently reversed the H23-induced inhibition of LTB4, 5-HETE, and 12-HETE production by PMN, suggesting inhibition at the phospholipase A2 (PLA2) level. Furthermore, addition of the cancer cell line Q9 inhibited 14C release from [14C]AA prelabeled PMN in a cell number-dependent manner in the buffer, with and without albumin. The supernatant of H23 cells also inhibited the production of LTB4 by PMN stimulated by A23187, as did the addition of H23 lysate or its 10(4) x g centrifugation supernatant. While neither the 10(5) x g supernatant (cytosol) nor the pellet (microsome) exhibited inhibitory activity, the combination of the separated cytosol and microsomal fractions restored the inhibitory activity. Furthermore, addition of the 10(4) x g supernatant of Q9 lysate to partially purified human cytosolic PLA2 inhibited PLA2 activity in a dose-dependent manner. Our results indicate that the lung cancer cell lines used in our study inhibit LTB4 production by human PMN through inhibition of phospholipase A2 activity, which may contribute to a predisposition to pulmonary infections in patients with lung cancer.

Evaluation of the effects of steroids on experimental septic lung injury.

To evaluate the clinical usefulness of steroids for septic lung injury, we investigated the effects of methylprednisolone (MP) on this disorder using an experimental rat model of cecal ligation and puncture (CLP). While 92% of the rats that underwent CLP (CLP rats) died within 30 h, those given high-dose MP (30 mg/kg) just after the operation (CLP + MP rats) survived for a significantly longer period (p < 0.01). Concentrations of endotoxin (ET) in arterial blood were significantly higher in the CLP + MP rats than in the CLP rats, while those in the bronchoalveolar lavage fluid (BALF) were significantly lower. Alveolar macrophages (AM) obtained from the CLP rats (CLP-AM) generated more O2-than did AM from sham-operated rats (sham-AM) following stimulation. However, the administration of MP did not reduce the upregulated generation of O2-by CLP-AM. While CLP-AM produced less leukotriene (LT)B4 than did sham-AM following stimulation with A23187, the administration of MP further reduced LTB4 production. When AM were cultured with [3H]arachidonic acid (3H-AA), the uptake of the isotope and the 3H release were significantly less in CLP-AM than in sham-AM. The administration of MP did not cause recoveries in the uptake and release of 3H-AA by CLP-AM. Although the survival time of CLP rats was significantly prolonged and the translocation of ET into BALF was reduced by steroid administration, the steroid effects were not explained by those on altered AM function. The upregulated generation of O2- and reduced LTB4 production from CLP-AM were not reversed by the treatment of this drug.

Modulatory effect of roxithromycin on human neutrophil function.

Neutrophils are thought to play a key role in tissue injury. We investigated the effect of roxithromycin, a 14-membered ring macrolide, on human neutrophil functions. The drug inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide (O2-) production and Ca2+ influx of human neutrophils. The inhibition was overcome by adding an inhibitor of cyclic AMP-dependent protein kinase (PKA), H-89. These results suggest that the drug affects O2- production and intracellular Ca2+ concentration of neutrophils via the action of PKA. Moreover, roxithromycin ameliorated endothelial cell injury induced by neutrophils, which may be, in part, due to the effect of the drug on neutrophils. Thus, roxithromycin may contribute to the treatment of diseases worsened by the excessive action of neutrophils.

Hyperresponsiveness of granulocytes to anaphylatoxins, C5a and C3a, in Churg-Strauss syndrome.

A 50-year-old man with Churg-Strauss syndrome showed granulocytes (GNLs) which generated more superoxide anion (O2-) than GNLs from healthy subjects following in vitro stimulation with C5a or C3a. Production of O2- subsided as the clinical symptoms improved with steroid treatment. A hyperresponsiveness of GNLs may be involved in this disorder.

[A case of post-traumatic arteriovenous fistula of the subclavian artery].

A 67-year-old woman was admitted to our hospital for a complete medical evaluation of a pulmonary nodule on a chest X-ray film, which was found during an annual check-up. A loud bruit was audible over the left chest and the supraclavicular region. Computerized tomography (CT) scan and magnetic resonance imaging revealed an arteriovenous fistula from a branch of the left subclavian artery into the paravertebral veins. Angiography disclosed that the arteriovenous fistula was fed by the left costocervical artery. Transarterial embolization of the arteriovenous fistula was done with 5 mechanical detachable coils and 2 micro coils. Postembolization angiography showed a marked decrease in blood flow and near-elimination of the arteriovenous fistula. The shrinkage of the arteriovenous fistula was also observed on CT scan. This patient had been in a traffic accident, and had been clinically well before the accident. Post-traumatic arteriovenous fistula was strongly suspected, because a pulmonary nodule on chest X-ray film and a bruit were found after a traffic accident.