Denaturing gradient gel electrophoresis analyses of microbial community from field-scale composter.

The diversity of microbial community during the decomposition of waste in a field-scale composter (Hazaka system) was investigated by denaturing gradient gel electrophoresis (DGGE). The composter operates at a high temperature through a self-heating system, creating a thermophilic (60-76 degrees C) stage during the initial phase and a mesophilic (45 degrees C) stage towards the later phase of the composting period. The pH of the system (pH 7.75-8.10) did not vary significantly during the process while moisture content was reduced from 48.8% to 25.1%. DGGE and 16S rDNA analyses showed that the following genera were found throughout the process: Propionibacterium sp., Methylobacterium sp., Pseudomonas sp., and Bradyrhizobium sp. Different Bacillus spp. thrive at the thermophilic or the mesophilic stage while Clostridium sp. was only found at the initial phase of the process. Staphylococcus sp. and Caulobacter sp. or Brevundimonas sp. existed during the later phase of the composting period.

Mechanism for bone invasion of oral cancer cells mediated by interleukin-6 in vitro and in vivo.

BACKGROUND: Osteoclastic bone resorption is an important step in bone invasion in several malignancies. Although interleukin (IL)-6 accelerates osteoclastic bone resorption, it remains unclear whether IL-6 may be involved in bone invasion of oral cancer. METHODS: The pit formation assay with calf femur-derived bone slices was performed to examine the bone-resorbing activity of osteoclasts and cancer cells. The chemotaxis activity of the culture media was analyzed by the use of Boyden chamber technique. Nude mice, which were inoculated with IL-6-producing oral cancer cells into masseter, were treated with anti-IL-6 neutralizing antibody, and mandibular-bone invasion of the cells was assessed. RESULTS: BHY, a bone-invasive oral cancer cell line, but not HNT, a noninvasive cell line, produced large amounts of IL-6. In a pit formation assay, addition of conditioned medium (CM) derived from BHY but not HNT increased osteoclastic bone resorption, and the effects were inhibited by anti-IL-6 antibody. BHY-secreted IL-6 showed significant chemotaxis activity for osteoclasts. Of note, CM from the cocultivation of osteoclasts and BHY markedly enhanced the cancer cell migration, and the chemotaxis activity was significantly reduced when anti-IL-6 antibody was added into the coculture and then CM were collected, but not when the antibody was added into the CM after they were collected. Furthermore, treatment with anti-IL-6 antibody almost completely inhibited mandibular bone invasion of BHY in nude mice. CONCLUSIONS: These results strongly suggest that IL-6 secreted by oral cancer cells plays a significant role in bone invasion.

Hyperexcitability of cervical motor neurons during neck flexion in patients with Hirayama disease.

We recorded the motor evoked potentials (MEPs) from the affected thenar muscles during neck flexion in 4 patients in the early progressive phase of Hirayama disease. The MEP size significantly increased during neck flexion in patients compared with normal controls. In 2 patients who were treated with a neck collar, the intrinsic muscle atrophy gradually recovered together with increased grip power, and the time course of changes in MEP during neck flexion became normal after treatment with a neck collar for 7-16 months. The technique described here would be useful for evaluating the vulnerability of cervical motor neurons and the therapeutic effect of the neck collar in Hirayama disease.

Role of osteopontin in bone remodeling caused by mechanical stress.

Changes in the number and proportion of osteopontin mRNA (Opn) expressing osteocytes and osteoclasts caused by the mechanical stress applied during experimental tooth movement were examined in the present study. Opn expression was detected in the osteocytes on the pressure side at the early stage, and gradually spread to those on the tension side and also to the osteoblasts and bone-lining cells in the alveolar bone. Only 3.3% of the osteocytes located on the pressure side expressed Opn in the interradicular septum of control rats; in contrast, the value was increased to 87.5% at 48 h after the initiation of tooth movement. These results indicate that these cells responded to mechanical stress loaded on the bone with expression of the osteopontin gene. Following the increased expression of Opn in these cells, a 17-fold greater number of osteoclasts compared with the control and numerous resorption pits were observed on the pressure side of the alveolar bone. Injection of arginine-glycine-aspartic acid-serine peptide but not that of arginine-glycine-glutamic acid-serine peptide strongly inhibited the increase in the number of osteoclasts. Furthermore, an in vitro migration assay demonstrated the chemotactic activity of osteopontin (OPN) on the precursor of osteoclasts. Our study strongly suggests that OPN is an important factor triggering bone remodeling caused by mechanical stress.

Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts.

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo.

Antibodies against Helicobacter pylori were detected in the cerebrospinal fluid obtained from patients with Guillain-Barré syndrome.

We examined the antibodies against Helicobacter pylori proteins in the cerebrospinal fluid (CSF) of 7 patients with Guillain-Barré syndrome (GBS). Crude H. pylori antigens, fractionated heat shock protein (HSP), and urease B (UB) from H. pylori antigens were separated by SDS-PAGE. With Western blot analysis, four of seven CSF samples had several IgG antibodies against H. pylori proteins, including HSP and UB. No cross reactivity against Campylobacter jejuni was observed. These antibodies may be involved in the immune responses of patients with GBS.

[A case of systemic lupus erythematosus developed with intestinal perforation].

A case of intestinal perforation associated with SLE is presented. A 54-year-old woman was diagnosed as having SLE twenty-five years ago when she had facial erythema, photosensitivity, oral aphtha, polyarthraliga, leukopenia, positive LE cell and positive antinuclear antibody. She had been treated with prednisolone and admitted to Kushiro City General Hospital because of one month history of fever and anorexia in February 1996. Laboratory findings did not reveal activity of SLE, and a diagnosis of urinary tract infection was made based on the findings of urinalysis. After severe diarrhea, disseminated intravascular coagulation (DIC) developed. A rectal perforation was revealed by endoscopic and radiological examination. An emergency laparotomy revealed necrosis of the rectum and sigmoidostomy was performed. The biopsied specimen of the rectum were diagnosed as gangrene of ischemic colitis histologically. Because of a penetration to the urinary bladder, an ureterocutaneostomy was performed. She died of sepsis and DIC on the 127th day of admission. Only 11 cases of intestinal perforation associated with SLE have been reported in Japan, and the association of vasculitis has been considered. In the present case, the prolonged use of prednisolone might cause the necrotizing ischemic colitis.

[A recovered case of SLE with central nervous system involvement who relapsed presenting new symptoms associated with development of serum anti-Sm antibody].

A rare SLE patient with central nervous system involvement (CNS-SLE) who relapsed presenting new symptoms associated with the development of serum anti-Sm antibody and was then successfully treated with cyclophosphamide (CY) pulse therapy is presented here. A 47-years old housewife was admitted to Kushiro City General Hospital because of fever, limb erythema and drowsy consciousness in September 1995. On the basis of convulsion, proteinuria, leukopenia, thrombopenia, serum positive tests for both anti-nuclear antibody and anti-SSA antibody and low complement levels, as well as elevations of IgG index and IL-6 in the cerebrospinal fluid (CSF), she was diagnosed as having CNS-SLE. Serum tests for anti CL-beta 2 GPI antibody and lupus anticoaglant was negative. Serum test for HBs antigen was positive. She was treated successfully with methylprednisolone (mPSL) pulse therapy and plasma exchange (PE). Prednisolone was gradually tapered to the dosage of 17.5 mg per day and she was discharged in April 1996. She was re-admitted because of fever, an exacerbation of skin eruption and arthralgia in October 1996. Serum anti-Sm antibody was found to be positive. mPSL pulse therapy was not effective. On the basis of hallucination and elevations of IgG index and IL-6 in the CSF, a diagnosis of relapsed CNS-SLE was made. However the level of IFN-alpha in the CSF was normal. Although PE was not effective, CY pulse therapy was markedly effective.

[Two cases of acetaminophen-induced pneumonitis].

This paper deals with two patients with acetaminophen-induced pneumonitis. A 64-year-old woman suffered from mastitis while being treated by corticosteroid therapy for phemphigoid. She was administered antibiotics and acetaminophen. However, her fever continued and she subsequently developed dyspnea and interstitial pneumonia. The other patient, a 70-year-old woman, was treated with corticosteroid for lower motor neuron disease. Anti-GM1-IgM antibodies were positive in her serum. She developed wet cough and mild fever. During treatment with antibiotics and acetaminophen, her illness was complicated by dyspnea and interstital pneumonia. As a result of histological findings of transbronchial lung biopsy specimens showing interstitial infiltration of mononuclear cells, as well as clinical courses in which cessation of acetaminophen directly lead to the improvement of interstitial pneumonia, both patients were diagnosed to have acetaminophen-induced pneumonitis. The peumonitis responded well to steroid therapy. In vitro culture of peripheral lymphocytes showed stimulated proliferation by acetaminophen in both patients. These findings suggest that allergic mechanism was involved in the pathogenesis of the pneumonitis. Underlying immunological disorders may have enhanced the occurrence. Although acetaminophen is one of the most popular drugs because of a very low incidence of side effects, this drug should be applied carefully, especially with patients who have such immunological disorders.

Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts.

Estrogen deficiency causes bone loss, which can be prevented by estrogen replacement therapy. Using a recently developed technique for isolation of highly purified mammalian osteoclasts, we showed that 17 beta-estradiol (E2) was able to directly inhibit osteoclastic bone resorption. At concentrations effective for inhibiting bone resorption, E2 also directly induced osteoclast apoptosis in a dose- and time-dependent manner. ICI164,384 and tamoxifen, as pure and partial antagonists, respectively, completely or partially blocked the effect of E2 on both inhibition of osteoclastic bone resorption and induction of osteoclast apoptosis. These data suggest that the protective effects of estrogen against postmenopausal osteoporosis are mediated in part by the direct induction of apoptosis of the bone-resorbing osteoclasts by an estrogen receptor- mediated mechanism.

Chick osteocyte-derived protein inhibits osteoclastic bone resorption.

In order to investigate the role of osteocytes in bone resorption, we examined the homogenate and conditioned medium from purified chick calvarial osteocytes in a pit-formation assay using unfractionated bone cells from mice. The osteocyte homogenate markedly inhibited pit formation, whereas the conditioned medium of osteocytes had no effect. This inhibitory activity was not the result of cytotoxicity of the homogenate. A novel bone-resorption-inhibitory protein was purified from collagenase-digested chick calvarial fragments enriched in osteocytes. The inhibitory protein, of molecular mass 18.5 kDa, showed significant dose-dependent inhibition of pit formation by unfractionated bone cells from mice and rabbits, and by human giant tumour cells. This protein also inhibited the bone-resorbing activity of purified osteoclasts in the pit-formation assay in the absence of other effector cells. Microinjection of the protein into osteoclasts caused disruption of the podosomes in the cells. The N-terminal 25-amino-acid sequence of the protein showed 68% identity to a part of Rho-GTP-dissociation inhibitor. Thus chick calvarial osteocytes may be involved in the regulation of bone resorption by osteoclasts.

Inhibitory mechanisms of H(+)-ATPase inhibitor bafilomycin A1 and carbonic anhydrase II inhibitor acetazolamide on experimental bone resorption.

The effects of the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (baf.A1) and the carbonic anhydrase II inhibitor acetazolamide (AZ) on bone resorption and procathepsin L secretion of rat osteoclasts were investigated using the bone slice assay method, pit formation test. Baf.A1 completely suppressed osteoclastic bone resorption stimulated by parathyroid hormone (PTH), but did not affect procathepsin L secretion, while AZ suppressed both bone resorption and procathepsin L secretion. These findings suggest that bone resorption by procathepsin L secretion and its processing are regulated by proton production and proton secretion.

Differentiation dependent expression of tensin and cortactin in chicken osteoclasts.

The expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg-laying hens maintained on a Ca-deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20-53 microns diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)-positive, bone resorbing, multinucleated syncytia in the presence of cytosine-1-beta-D-arabinofuranoside in a time dependent manner (day 1-6). Differentiation into osteoclast-like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP-DH control levels, tensin and cortactin mRNA levels increased by 7- and 10-fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6- and 15-fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast-like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast-like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast-like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteoclast precursors.

Regional blood flow distribution and left ventricular output during early neonatal life: a quantitative ultrasonographic assessment.

To examine the serial changes of left ventricular output and regional blood flow distribution during the early neonatal period, we measured blood flow volume in the ascending aorta, middle cerebral artery, celiac artery, superior mesenteric artery, and renal artery in 23 normal term infants at 1, 4-8, 24, and 96 h after birth. The blood flow volume in each vessel was measured by the pulsed Doppler technique. In the middle cerebral artery, celiac artery, and superior mesenteric artery, the blood flow volume at 1 and 4-8 h of age was significantly lower than after 24 h of age. In contrast, renal artery blood flow volume did not change significantly throughout the study period. The reduced organ blood flow volume soon after birth was related to a low diastolic blood flow in the major vessels, and the percent diastolic integral of blood flow velocity in each vessel showed an inverse linear correlation with the diameter of the ductus arteriosus. The left ventricular output 1 h after birth was 365 +/- 69 mL/kg/min, which was significantly higher than after 4-8 h of age. Left ventricular output gradually declined to 301 +/- 63 mL/kg/min at 4-8 h of age (p < 0.05 versus 96 h), 272 +/- 48 mL/kg/min at 24 h, and 258 +/- 54 mL/kg/min at 96 h. There was a significant positive correlation between left ventricular output and the ductus arteriosus diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in pulmonary venous flow pattern during early neonatal life.

OBJECTIVE: To investigate serial changes in the pattern of flow in the pulmonary vein during the early neonatal period. METHODS: Pulsed Doppler echocardiography was used to measure flow in the right upper pulmonary vein in 26 normal newborn infants. Peak flow velocity during systole (S) and diastole (D) and flow velocity at indents between the systolic and diastolic fraction (O) and between the diastolic and systolic fraction (X) were measured 1, 4-8, 24, and 96 hours after birth. The heart rate and diameter of the ductus arteriosus were measured simultaneously. RESULTS: Continuous and phasic high flow velocity waveforms were seen 1 and 4-8 hours after birth. The mean (SD) peak flow velocities of X, S, O, and D an hour after birth were 35.2 (13.6) cm/s, 73.1 (23.1) cm/s, 58.5 (20.5) cm/s, and 81.5 (19.2) cm/s respectively. There were significant decreases in X, S, O, and D by 24 hours of age (p < 0.01 v 1 hour after birth) to 8.1 (10.3) cm/s, 52.8 (18.0) cm/s, 38.6 (14.5) cm/s, and 54.4 (11.2) cm/s respectively. These results indicate intermittent flow in the pulmonary vein, with flow stopping between diastole and systole. These flow velocities, X, S, O, and D, correlated well with the diameter of the ductus arteriosus (r = 0.80 v X, r = 0.62 v S, r = 0.63 v O, r = 0.75 v D). CONCLUSION: This serial study showed changes in normal pulmonary vein flow patterns during the early neonatal period. The continuous and high flow velocity waveform that was seen immediately after birth resembled the pattern of pulmonary vein flow seen in congenital pulmonary stenosis and in cases of acute volume overload. This waveform may reflect a sudden increase in pulmonary circulatory volume with additional left to right shunting through the ductus arteriosus in relatively hypoplastic pulmonary veins.

Moebius syndrome: continuous tachypnea verified by a polygraphic study.

Four polygraphic recordings were obtained in three cases of Moebius syndrome. The cases were a 4-month-old girl (Case 1), a 4-year-old boy (Case 2), and a 5-year-old girl (Case 3). The recordings revealed that in all three cases there was a lack of rapid eye movements to the lateral side, continuous low amplitude on chin EMG, and continuous tachypnea. In Case 3, polygrams were recorded at 2 and 5 years of age, both records showing the same pattern of tachypnea. Arterial blood gas analysis (AGA), PH, PaCO2 and PaO2 were found to be within normal limits. It has been assumed that this syndrome includes brainstem dysplasia, according to the chief symptoms, and autopsy and auditory brainstem response (ABR) findings. ABR examination showed low amplitude of waves, a prolonged wave I-V interval, and the absence of wave V. It is known that there is a close relationship between this syndrome and respiratory disorders. But there have only been a few reports on respiratory disorders in this syndrome. It is strongly believed that continuous tachypnea is an important symptom of Moebius syndrome.

[A case of spontaneous pneumothorax due to rupture of bleb infected with aspergillosis].

A 17-year-old male was diagnosed as having left spontaneous pneumothorax by chest X-ray. Chest CT scan showed a cystic shadow with a thick wall in the left apex. Thoracotomy was performed, and the bleb was resected. Histologically, aspergillus was found in the cavity and the wall of the resected bleb. In this case, we consider that focal aspergillus infection in the bleb caused spontaneous pneumothorax. In cases of spontaneous pneumothorax in which the bleb wall is thick, the bleb should be evaluated by histological and bacteriological examination. Such a case has been rarely reported in Japan.

A simple method to assess osteoclast-mediated bone resorption using unfractionated bone cells.

To determine osteoclastic bone resorption we established a simple assay system in which unfractionated cells obtained from femora of 13-day-old mice were cultured on a dentine slice and the number of osteoclasts and their induced pit area on the slices were measured. When the bone cells (1 x 10(5) cells/dentine slice) were cultured in the presence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or human parathyroid hormone (hPTH) for 4 days, at which time newly-formed osteoclasts were not detected, the pit area was dose-dependently increased, being a 4.3- or 4.1-fold respective increase over the control at a 10(-8) M concentration of hormones. Chick calcitonin (cCT) inhibited the osteoclastic bone resorption induced by either of these hormones. cCT alone also suppressed the bone resorption by the cells (3 x 10(5) cells/dentine slice). These findings indicate that 1,25(OH)2D3 or hPTH may mainly activate pre-existing osteoclasts, resulting in increased bone resorption, and that cCT may suppress this osteoclastic activity. When 1,25(OH)2D3 or hPTH was added to the cells pre-cultured in factor-free medium for 6 days, at which time pre-existing osteoclasts had almost degenerated, new osteoclasts were formed, resulting in an increase in pit formation. Thus this system is a useful method which could more sensitively evaluate the effects of hormones or factors on osteoclast formation and activation than other previous systems.

Hospital-onset tuberculosis in compromised host.

A total of 21 patients developed active tuberculosis (TB) during hospitalization. Active TB was identified by bacteria-positive, biopsy or autopsy. Infection was confirmed to the lung, pleura, lymph node and miliary lesions and 7 patients had open tuberculosis. In half of the patients, chest X-ray films demonstrated unusual findings in adult tuberculosis: lower lung field pneumonia and miliary pattern. All the patients suffered from severe underlying diseases and an intensive therapy with steroid, immunosuppressive agents, antitumor drugs, radiation and operation was found as predisposing factors for TB occurrence. Nine patients recovered from current infection with anti-tuberculosis drugs; 14 patients died and TB directly caused death in 8 patients. These data strongly suggest that TB is one of the most important infections in compromised hosts. We emphasize that this infection presents a serious clinical problem in a general hospital today.

[A case of Wegener's granulomatosis with a giant solitary pulmonary mass].

A 44-year-old woman was admitted to hospital with a history of high fever, cough, and weight loss. Her chest X-ray film showed a giant solitary pulmonary mass in the left lower lobe. After admission, she developed bilateral eyeache and left exophthalmos. CT film of the head showed a massive lesion in the sinuses. Biopsy specimens obtained from both massive lesions showed either epithelioid granulomas with Langhans' giant cells or necrotising vasculitis. Antineutrophil cytoplasmic antibodies, which have high sensitivity and specificity for active WG, were found. The diagnosis of WG was thus made.