Comparative studies of suppression of malignant cancer cell phenotype by antisense oligo DNA and small interfering RNA.

One of the distinguishing features of malignant tumor cells is the ability to proliferate in an anchorage-independent manner; methods that effectively suppress this phenotype may be applicable to the therapeutic inhibition of the malignancy of cancers. Interfering RNA is a potentially powerful tool for cancer therapy because of its specificity of target selection and remarkably high efficiency in target mRNA suppression. We studied the use of two knockdown strategies, antisense oligo DNA (AS-ODN) and small interfering RNA (siRNA), and showed how the anchorage-independent proliferation of malignant cells could be blocked efficiently. Anchorage-independent proliferation of rat fibroblasts transformed with v-src was suppressed with only a single 1-microM dose of AS-ODN; similar suppression using siRNA required treatment with 1 nM siRNA every 12 h. With our experimental system, the molecular stability of AS-ODN allowed the use of a simple treatment regimen to control the amount of the target molecule, providing that the treatment dose was sufficiently high. In comparison, siRNA treatment was effective at lower doses, but more frequent treatment was necessary to achieve the same suppression of proliferation.

Evolving a lingua franca and associated software infrastructure for computational systems biology: the Systems Biology Markup Language (SBML) project.

Biologists are increasingly recognising that computational modelling is crucial for making sense of the vast quantities of complex experimental data that are now being collected. The systems biology field needs agreed-upon information standards if models are to be shared, evaluated and developed cooperatively. Over the last four years, our team has been developing the Systems Biology Markup Language (SBML) in collaboration with an international community of modellers and software developers. SBML has become a de facto standard format for representing formal, quantitative and qualitative models at the level of biochemical reactions and regulatory networks. In this article, we summarise the current and upcoming versions of SBML and our efforts at developing software infrastructure for supporting and broadening its use. We also provide a brief overview of the many SBML-compatible software tools available today.

Abnormal development of serotonin nerve fibers in the visual cortex in rats with methylazoxymethanol-induced microcephaly.

The postnatal development of serotonin (5HT)-immunoreactive axons was studied in the visual cortex of the cerebrum in both normal and microcephalic rats during early postnatal and young adult stages. Severe microcephaly in rat offspring was induced by prenatal exposure to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation. From postnatal day 1 (PND 1) to PND 5, fine and short 5HT fibers were irregularly dispersed throughout the occipital cortex in both the control and MAM-treated rats (MAM-rats). A conspicuous aggregation of dot-like 5HT terminals was found in controls, but not in MAM-rats, in a shallow layer of the dorsomedial region of the occipital cortical plate. On PND 7, such an aggregation of 5HT terminals was found in both groups. The density of the aggregation increased up to PND 9, but then decreased gradually, finally becoming unrecognizable at around PND 15 in both groups. MAM-rats, however, always showed hyperaggregation of 5HT terminals when compared with controls on the same PND. The density of 5HT fibers gradually increased, and finally made up a network-like formation at PND 28 in both groups, its pattern was essentially identical to the abnormal distribution of 5HT fibers during the later stage. As a result, the network-like formation of 5HT fibers in the MAM-rats at PND 28 was markedly twisted and somewhat hyperdense. In Nissl-stained preparations from PND 9 to 15, the 5HT terminal aggregation in the control rats was precisely confined to the newly forming layer IV of the visual cortex. In the MAM-rats, on the other hand, the aggregation of 5HT terminals was not associated with a specific cortical layer because of a disarranged cytoarchitecture of the microcephaly.

Cortical fiber distribution in the somatosensory cortex of rats following prenatal exposure to X-irradiation.

Exposure of fetal rats to X-irradiation at a dose of 1.5 Gy on gestational day 15 results in severe microcephaly. In these X-irradiated rats with microcephaly, a possible alteration of local circuits in the somatosensory cortex was examined using a fluorescent dye, DiI. The dye was placed at the superficial part of the transversely sectioned somatosensory cortex just beneath the pial surface of both the control and X-irradiated rats on postnatal day 11. In the controls, DiI-labeled fibers showed a layer-dependent distribution in the somatosensory cortex, whereas in the X-irradiated rats, this layer-dependent distribution of DiI-labeled fibers was distorted. Furthermore, the fasciculus observed beneath cortical layer VI (white matter), which was made of DiI-labeled axons originating from the cortical neurons, was well developed in the controls. On the other hand, in the X-irradiated rats, the fasciculus made of DiI-labeled fibers was split, reduced in number, and irregularly distributed. These observation suggest that prenatal exposure of fetal rats to X-irradiation may affect the development of local circuits in the cerebral cortex, and may also damage the axonal projection of cortical neurons to the thalamus.

Association of increased prostaglandin E2 content in bronchoalveolar lavage fluid and intrathoracic malignancy.

Prostaglandin E2 (PGE2), a metabolite of arachidonic acid, plays an important role in immunoregulation. We have previously reported an increased PGE2 content in bronchoalveolar lavage fluid (BALF) in 18 patients who had primary lung cancer as compared with 6 patients who had benign lung diseases and 10 normal smokers. In this article, we report our further observation, including additional 73 patients with primary lung cancer, 11 patients who had carcinoma other than lung cancer with and without lung metastases, and 24 patients with various benign lung diseases. Prostaglandin E2 content of BALF in patients with primary lung cancer and patients with extrathoracic cancer with lung metastases was markedly increased to 283.7 +/- 423.8 and 278.3 +/- 303.4 pg/ml, respectively. In contrast, patients who had extrathoracic cancer without lung metastases and patients with benign lung diseases had PGE2 content in BALF of 22.6 +/- 6.6 and 27.2 +/- 12.4 pg/ml, respectively. Values between the subgroups of patients with lung cancer (primary and metastatic) and patients with benign diseases or extrathoracic cancer without lung metastasis were significantly different (p < .001). Follow up of eight patients who successfully underwent surgical resection of cancer showed decreased levels of PGE2 in all but two patients. Although the exact cause and mechanism of the observed increase of PGE2 in BALF of patients with lung cancer are not clear at this point, current study together with our previous observations clearly indicates that the presence of lung cancer, both primary and metastatic, is associated with an elevated PGE2 content of BALF. The clinical significance of this observation is not known. However, an increased amount of PGE2 in BALF might adversely affect the course of lung cancer, because PGE2 is a down regulator of immune response against infection and neoplasm. Further investigations into the mechanism(s) and the role of PGE2 in BALF are warranted.

The value of the forced expiratory time in the physical diagnosis of obstructive airways disease.

OBJECTIVE: To evaluate the test characteristics of the forced expiratory time (FET) in the diagnosis of obstructive airways disease. DESIGN: A cross-sectional diagnostic test study. The FET of 400 subjects was measured by a physician examiner and was compared with the criterion standard of spirometry. In a second sample of 100 subjects, the FET was measured by pairs of physician examiners to evaluate interexaminer agreement. SETTING: A pulmonary function test laboratory at a tertiary care hospital that receives referrals for preoperative evaluations, acute and chronic pulmonary disease, and occupational lung disease. SUBJECTS: A consecutive sample of patients who were referred to the pulmonary function laboratory from primary care internists, pulmonary physicians, and surgeons. INTERVENTIONS: None. MAIN OUTCOME MEASURES: The sensitivity and specificity of the FET in the diagnosis of obstructive airways disease at cutoff values ranging from 2 to 14 seconds. A receiver operating characteristic curve was used to evaluate the diagnostic performance of the FET. Likelihood ratio lines were determined using a logistic regression model adjusting for the subjects age. Interexaminer agreement was evaluated with a kappa statistic. RESULTS: Using the FET maneuver with a cutoff value of 6 seconds will correctly diagnose the greatest number of subjects with obstructive airways disease. The FET maneuver is more discriminating for subjects 60 years or older compared with younger subjects. The positive likelihood ratio for a subject aged 60 years or older with an FET of 4 to 6 seconds is 0.42 (95% confidence interval [CI], 0.24 to 0.73); of 6 to 8 seconds, 2.19 (95% CI, 1.02 to 4.80); and of greater than 8 seconds, 4.08 (95% CI, 2.54 to 6.79). The kappa statistic for interexaminer agreement is 0.70. CONCLUSIONS: The FET demonstrates moderately good performance as a diagnostic test for obstructive airways disease. The value of the test will depend on the pretest probability of disease and the clinical circumstances in which it is used.

Gamma-radiation produces abnormal Bergmann fibers and ectopic granule cells in mouse cerebellar cortex.

Morphological changes in Bergmann glial fibers in the developing cerebellar cortex produced by exposure to gamma-rays were investigated in association with ectopic granule cells. Six-day-old mice that had been exposed to 3 Gy of gamma-radiation were killed 6 hours after exposure or at 7 through 30 days of age. Their cerebella were examined histologically and immunohistochemically for glial fibrillary acidic protein in Bergmann fibers. Extensive cell death took place in the external granular layer (EGL) of the cerebellum from 6 through 24 hours after exposure. This led to the thinning of the EGL and a decrease in the number of migrating cells in the molecular layer. The number of Bergmann cells was not decreased, but the fibers in the molecular layer were distorted; whereas, in the control these fibers were straight and perpendicular to the pial surface. The EGL began to recover 2 days after exposure, and abnormally oriented migrating cells were seen. At 17 days of age, some cell clustering was observed in the molecular layer of the irradiated cerebellum. Distortion of the Bergmann fibers was marked in regions where ectopic granule cells appeared at 30 days of age. These findings suggest that the distortion of Bergmann fibers leads to the production of ectopic granule cells after exposure to gamma-radiation.

Developmental alteration of serotonin neurons in the raphe nucleus of rats with methylazoxymethanol-induced microcephaly.

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM), an anti-mitotic agent, on day 15 of gestation induces severe microcephaly in the offspring. The present study first investigated a developmental alteration of serotonin (5HT) neurons immunohistochemically in the dorsal and median raphe nuclei in serial sections in both control and microcephalic rats (MAM-rats) at 35 days of age. 5HT-immunoreactive neurons in the MAM-rats were reduced in number and irregularly distributed in the dorsal and median raphe nuclei compared with those in the control. The dendrites of neurons in these nuclei in the MAM-rats were very short and twisted. A follow-up observation on the development of the cerebral cortex at 5, 9 and 28 days of age was performed using Nissl-stained preparations, which revealed a disorganized cell arrangement in the cerebral cortex of the MAM-rats at the very early postnatal period. Furthermore, the distribution of 5HT-immunoreactive fibers into the cerebral cortex was also examined using brains of 28 days of age. In MAM-rats of this age, abnormally tortuous 5HT-immunoreactive fibers were observed in the cerebral cortex. 5HT neurons in the raphe nuclei are known to project their ascending axons widely into the entire cortical area during the 1st postnatal week. Thus, the association of disorganized cortical cell arrangement and the hyperdense and tortuous distribution of 5HT-immunoreactive fibers in the cerebral cortex support the idea of target-dependent secondary degeneration of 5HT neurons in the dorsal and median raphe nuclei of the MAM-rats.

Phenotype and function of natural killer cells in patients with bronchogenic carcinoma.

Decreased peripheral blood natural killer (NK) cell lytic activity may be associated with tumor presence. We evaluated peripheral blood NK lytic activity in 38 patients with bronchogenic carcinoma and compared this to ten normal volunteers of comparable age. The patients with carcinoma had significantly (P less than 0.001) less NK activity (20 +/- 17 lytic units at 25% specific lysis)/10(6) peripheral blood lymphocytes) against the K562 tumor target compared to normal (69 +/- 9, SD). NK subpopulations can be defined phenotypically using CD56, CD16, and CD3 monoclonal antibodies and express differing degrees of lytic activity. NK cells from patients with carcinoma had the same absolute number of CD56+ cells and percentage of CD56+CD16+ NK cells (the most lytic subpopulation). However, patients with carcinoma had significantly (P less than 0.001) more CD56+CD16-CD3- cells in their overall NK population. This is of note, since this subpopulation is the least lytic. Patient NK cells bound to tumor cells as effectively as those from normal volunteers; however, the maximum rate of kill of patient NK cells was significantly (P less than 0.001) less. Thus, decreased NK lytic activity in patients with carcinoma was not due to decreased numbers or proportion of NK cells in peripheral blood or to defective tumor cell binding, but rather to the large CD56+CD16-CD3-NK subpopulation which is characterized by minimal lytic activity. The relation of this NK cell population with the presence of carcinoma is currently under investigation.

Lymphokine-activated killer cell activity in lung cancer.

This study evaluates local pulmonary immune effector cell lytic activity. Purified lymphocyte populations were isolated from BALF obtained from 18 patients with bronchogenic carcinoma, six patients with lung disorders other than cancer, and ten normal control volunteers matched for age and smoking history. These cells were evaluated for NK and LAK cell lytic activity against NK-resistant LAK-sensitive tumor targets (A549 pulmonary tumor and Daudi tumor cells) and an NK-sensitive tumor (K562); LAK activity was detected in BALF from 6 of the 18 patients with cancer. The remaining patients with cancer, the subjects with pulmonary disease other than cancer, and the normal volunteers had no detectable lytic activity. Peripheral blood lymphocytes from all subjects had only NK lytic activity and did not kill the pulmonary tumor target; AMs were not tumoricidal. Interleukin-2, which is required for LAK cell activation, was detected only in BALF recovered from the six patients with pulmonary LAK lytic activity. These results demonstrate that activated LAK cells, capable of killing pulmonary tumor cells, are present in BALF of some patients with bronchogenic carcinoma. This lytic LAK cell population represents a local pulmonary response against the lung cancer in the absence of systemic tumoricidal activity. The functional status of pulmonary immune effector cells, as well as the type and quantities of cytokines in the lung determine local responsiveness to bronchogenic carcinoma and may well control the course of this disease.

Elevated prostaglandin E2 levels in bronchoalveolar lavage fluid of patients with bronchogenic carcinoma.

Understanding local pulmonary immunoregulatory mechanism(s) in patients with carcinoma of the lung is an important step towards the development of innovative methods of treatment. Prostaglandin E2 plays an integral role in immunoregulation. Therefore, we evaluated PGE2 concentrations in BALF from 18 patients with bronchogenic carcinoma, compared to that from six patients with pulmonary diseases other than carcinoma and ten normal smokers of similar age. The level of PGE2 in patients with lung carcinoma (158.1 +/- 88.7 pg/ml) was significantly (p less than 0.001) higher than the other two groups (16.2 +/- 6.9 and 4.4 +/- 3.4 pg/ml). Levels of PGE2 also varied among patients with carcinoma of different cell types. Patients with SQCA had significantly (p less than 0.001) higher levels of PGE2 (242.7 +/- 29.4 pg/ml) than patients with ADCA or SCCA (82.3 +/- 27.9 and 66.3 +/- 15.2 pg/ml, respectively). Furthermore, there was a marked difference in PGE2 concentration between carcinomatous lung and clinically noninvolved lung in patients with SQCA and ADCA. Further study is warranted to determine the interactions between PGE2 and other cytokines (interleukin-1, IL-2, and tumor necrosis factor), as well as the activity of cytolytic lymphocytes (LAK cells) in the lungs of patients with bronchogenic carcinoma.

Ventilatory response of spinal cord-lesioned subjects to electrically induced exercise.

Seven human spinal cord-lesioned subjects (SPL) underwent electrically induced muscle contractions (EMC) of the quadriceps and hamstring muscles for 10 min: 5 min control, 2 min with venous return from the legs occluded, and 3 min postocclusion. Group mean changes in CO2 output compared with rest were +107 +/- 30.6, +21 +/- 25.7, and +192 +/- 37.0 (SE) ml/min during preocclusion, occlusion, and postocclusion EMC, respectively. Mean arterial CO2 partial pressure (PaCO2) obtained from catheterized radial arteries at 15- to 30-s intervals showed a significant (P less than 0.05) hypocapnia (36.2 Torr) during occlusion and a significant (P less than 0.05) hypercapnia (38.1 Torr) postocclusion relative to a group mean preocclusion EMC PaCO2 of 37.5 Torr. Relative to preocclusion EMC, expired ventilation (VE) decreased during occlusion and increased after release of occlusion. However, changes in VE always occurred after changes in end-tidal PCO2 (mean 41 s after occlusion and 10 s after release of occlusion). In the two subjects investigated during hyperoxia, the VE and PaCO2 responses to occlusion and release did not differ from normoxia. We conclude that the data do not support mediation of the EMC hyperpnea in SPL by humoral mechanisms that others have proposed for mediation of the exercise hyperpnea in spinal cord-intact humans.

Welders' pneumoconiosis: tissue elemental microanalysis by energy dispersive x ray analysis.

Histological examination on lung tissue obtained from 10 symptomatic welders was performed by two certified pathologists without the knowledge of the patients' clinical condition. In all cases, there was some degree of interstitial fibrosis; in five the degree of fibrosis was considered to be moderate to pronounced. The tissue was also analysed by energy dispersive x ray analysis and elemental contents were compared with age matched controls. There was a large amount of iron in the lungs of welders but the silicon content did not differ from the control subjects. No specific foreign element was detected. It is concluded that (1) interstitial pulmonary fibrosis is seen in some welders and (2) the cause of fibrosis does not appear to be coexisting silicosis.

Ventilatory and PaCO2 responses to voluntary and electrically induced leg exercise.

We studied the role of central command mediation of exercise hyperpnea by comparing the ventilatory and arterial CO2 partial pressure (PaCO2) responses to voluntary (ExV) and electrically induced (ExE) muscle contractions in normal, awake human subjects. We hypothesized that if central command signals are critical to a normal ventilatory response, then ExE should cause a slower ventilatory response resulting in hypercapnia at the onset of exercise. ExE was induced through surface electrodes placed over the quadriceps and hamstring muscles. ExE and ExV produced leg extension (40/min) against a spring load that increased CO2 production (VCO2) 100-1,000 ml/min above resting level. PaCO2 and arterial pH during work transitions and in the steady state did not differ significantly from rest (P greater than 0.05) or between ExE and ExV. The temporal pattern of ventilation, tidal volume, breathing frequency, and inspired and expired times, and the ventilation-VCO2 relationship were similar between ExE and ExV. We conclude that since central command was reduced and/or eliminated by ExE, central command is not requisite for the precise matching of alveolar ventilation to increases in VCO2 during low-intensity muscle contractions.

Is the hyperpnea of muscular contractions critically dependent on spinal afferents?

We studied the role of spinal afferent pathways in the hyperpnea of electrically induced muscle contractions (ExE). The ventilatory (VE) and arterial CO2 partial pressure (PaCO2) responses were measured at rest and during two levels of ExE in awake human paraplegic subjects with clinically complete lesions of the spinal cord (range T4-T11). We hypothesized that if peripheral neural drive is critical to a normal ventilatory response, then ExE in the absence of intact pathways should cause a lower ventilatory response resulting in hypercapnia at the onset of ExE. ExE was induced by stimulation of the quadriceps and hamstring muscles that approximately doubled the resting level of CO2 production (VCO2). PaCO2 during work transitions and in the latter stages of ExE did not differ significantly from that at rest. Arterial pH progressively declined over time during ExE (P less than 0.01) as a result of increased lactate concentration (P less than 0.01). The linear relationship between VE and VCO2 was similar to that found for normal human subjects during ExE (P = 0.73). These data suggest that VE and presumably alveolar ventilation (VA) can be appropriately matched to VCO2 during low-intensity muscle contractions of the lower extremities in the absence of intact spinal afferent pathways. Moreover, since it is unlikely that postulated "central command" mechanisms were initiated during ExE in these paraplegic subjects, the data provide support for our previous conclusion that central command is not obligatory for matching VA to VCO2 (J. Appl. Physiol. 64: 218-225, 1988).

Temporal pattern of arterial CO2 partial pressure during exercise in humans.

The major objective of this study was to test the hypothesis that arterial CO2 partial pressure (PaCO2) does not change in transitions from rest to steady-state exercise and between two levels of exercise. Nine young adults exercised on a treadmill or a bicycle (sit or supine) for 5 min at a mild work load (heart rate = 90 beats X min-1) and then 3 min at a moderate work load (heart rate = 150 beats X min-1). In some studies the moderate work load preceded the mild work load. Arterial blood was sampled from a catheterized artery. During all exercise tasks isocapnia was not strictly maintained (F greater than 4.0, P less than 0.001). For example, a 1-to 2-Torr hypocapnia was the dominant trend during the first 15-45 s after increasing treadmill speed, and a transient hypercapnia was most prevalent when treadmill speed was decreased. During steady-state exercise PaCO2 did not deviate by more than 1-3 Torr from PaCO2 during any resting posture, and PaCO2 differences between exercise intensities and conditions did not exceed 1-2 Torr. A mouthpiece-breathing valve system was not used in most studies, but when this system was used, it did not consistently affect exercise PaCO2. Increasing inspired O2 to 40% likewise did not consistently alter exercise PaCO2. Failure to maintain isocapnia throughout exercise indicates that the matching of alveolar ventilation (VA) to lung CO2 delivery is not exquisitely precise. Accordingly it is inappropriate to base theories of the exercise hyperpnea on the heretofore contention of precise matching.(ABSTRACT TRUNCATED AT 250 WORDS)