Effects of delta-opioid receptor stimulation and inhibition on hippocampal survival in a rat model of forebrain ischaemia.

BACKGROUND: It has been reported that delta-opioid (DOP) receptor agonists may be neuroprotective in the central nervous system. However, the DOP agonist [d-Ala(2), d-Leu(5)]enkephalin (DADLE) does not produce neuroprotection in severe forebrain ischaemia. The aim of this study was to examine the effects of DADLE on hippocampal neurone survival against less severe forebrain ischaemia. METHODS: Intraperitoneal injection of DADLE (0 or 16 mg kg(-1)) in male Sprague-Dawley rats was performed 30 min before ischaemia. Severe (10 min), moderate (8 min), or mild (6 min) forebrain ischaemia was produced by bilateral carotid occlusion combined with hypotension (35 mm Hg) under isoflurane (1.5%) anaesthesia. Naltrindole (10 mg kg(-1)) (DOP antagonist) was administered 30 min before DADLE in order to confirm DOP receptor activation in the neuroprotective efficacy of DADLE. Naltrindole alone was also administered 30 min before ischaemia to examine endogenous DOP agonism as a self-protecting mechanism against ischaemia. All animals were evaluated neurologically and histologically after a 1 week recovery period. RESULTS: DADLE improved neurone survival in hippocampal CA3 and dentate gyrus (DG) sectors. CA1 neurones were not protected against moderate and mild ischaemia. Naltrindole abolished DADLE neuroprotection in the CA3 and DG after both moderate and mild ischaemia. Interestingly, regardless of co-administration of DADLE, naltrindole significantly worsened neuronal injury in the CA1 region after mild ischaemia. CONCLUSIONS: These results suggest that DADLE provides limited neuroprotection to relatively ischaemia-resistant regions but not to selectively vulnerable regions. This was probably mediated by DOP stimulation. Pre-ischaemic treatment with a DOP antagonist, regardless of co-administration of DADLE, worsened neuronal damage at the selectively vulnerable regions only after mild forebrain ischaemia. These data suggest that DOP activation with endogenous DOP ligand may be involved in self-protecting ischaemia-sensitive regions of the brain.

Cerebroside elicitors found in diverse phytopathogens activate defense responses in rice plants.

Cerebrosides A and C, compounds categorized as glycosphingolipids, were isolated in our previous study from the rice blast fungus (Magnaporthe grisea) as novel elicitors which induce the synthesis of rice phytoalexins. In this paper, these cerebroside elicitors showed phytoalexin-inducing activity when applied to plants by spray treatment and also induced the expression of pathogenesis-related (PR) proteins in rice leaves. This elicitor activity of the cerebrosides showed the structural specificity as that for the induction of phytoalexins. Ceramides prepared from the cerebrosides by removal of glucose also showed the elicitor activity even in lower level compared to the cerebrosides. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus, an economically devastating agent of disease of rice in Japan. The cerebrosides elicitors protected rice plants from other disease as well and were found to occur in a wide range of different phytopathogens, indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions.

Craniosacral rhythm: reliability and relationships with cardiac and respiratory rates.

Craniosacral rhythm (CSR) has long been the subject of debate, both over its existence and its use as a therapeutic tool in evaluation and treatment. Origins of this rhythm are unknown, and palpatory findings lack scientific support. The purpose of this study was to determine the intra- and inter-examiner reliabilities of the palpation of the rate of the CSR and the relationship between the rate of the CSR and the heart or respiratory rates of subjects and examiners. The rates of the CSR of 40 healthy adults were palpated twice by each of two examiners. The heart and respiratory rates of the examiners and the subjects were recorded while the rates of the subjects' CSR were palpated by the examiners. Intraclass correlation coefficients were calculated to determine the intra- and inter-examiner reliabilities of the palpation. Two multiple regression analyses, one for each examiner, were conducted to analyze the relationships between the rate of the CSR and the heart and respiratory rates of the subjects and the examiners. The intraexaminer reliability coefficients were 0.78 for examiner A and 0.83 for examiner B, and the interexaminer reliability coefficient was 0.22. The result of the multiple regression analysis for examiner A was R = 0.46 and adjusted R2 = 0.12 (p = 0.078) and for examiner B was R = 0.63 and adjusted R2 = 0.32 (p = 0.001). The highest bivariate correlation was found between the CSR and the subject's heart rate (r = 0.30) for examiner A and between the CSR and the examiner's heart rate (r = 0.42) for examiner B. The results indicated that a single examiner may be able to palpate the rate of the CSR consistently, if that is what we truly measured. It is possible that the perception of CSR is illusory. The rate of the CSR palpated by two examiners is not consistent. The results of the regression analysis of one examiner offered no validation to those of the other. It appears that a subject's CSR is not related to the heart or respiratory rates of the subject or the examiner.

The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1.

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1, is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF-induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

Efficacy of CS-834 against experimental pneumonia caused by penicillin-susceptible and -resistant Streptococcus pneumoniae in mice.

The efficacy of CS-834, a novel oral carbapenem, was assessed by using a murine model of pneumonia caused by penicillin-susceptible and penicillin-resistant Streptococcus pneumoniae and was compared with those of oral cephems, i.e., cefteram pivoxil, cefpodoxime proxetil, cefdinir, and cefditoren pivoxil. Intranasal inoculation of 10(6) CFU of penicillin-susceptible or penicillin-resistant S. pneumoniae in the exponential growth phase induced pneumonia and bacteremia in ddY mice within 48 h. For the treatment of infections caused by the penicillin-susceptible strain the antibiotics were administered orally at 0.4, 2, and 10 mg/kg of body weight twice daily for 2 days beginning at 24 h after bacterial inoculation, and for the treatment of infections caused by a penicillin-resistant strain the antibiotics were administered at 2, 10, and 50 mg/kg twice daily for 2 days beginning at 24 h after bacterial inoculation. Among the antibiotics tested, CS-834 exhibited the most potent efficacy against both types of strains. Against infections caused by penicillin-susceptible S. pneumoniae, CS-834 at all doses significantly reduced the numbers of viable cells in both the lungs and blood. Cefpodoxime proxetil at all doses and cefteram pivoxil and cefditoren pivoxil at doses of 2 and 10 mg/kg showed comparable efficacies. Against infections caused by penicillin-resistant S. pneumoniae, CS-834 at doses of 10 and 50 mg/kg showed the most potent efficacy among the antibiotics tested, resulting in the maximum decrease in the numbers of viable cells in the lungs. Comparable efficacies were observed with cefteram pivoxil and cefpodoxime proxetil at doses of 50 mg/kg each. The concentration of CS-834 in the lungs and blood was higher than that of cefdinir and was lower than those of the other antibiotics tested, suggesting that the potent therapeutic efficacy of CS-834 reflects its strong activity against S. pneumoniae.

Diagnostic value of technetium-99m methoxyisobutyl isonitrile (99mTc-MIBI) scintigraphy in detecting thyroid cancer metastases: a critical evaluation.

Technetium-99m methoxyisobutyl isonitrile (99mTc-MIBI) scintigraphy has recently been used in clinical application for detecting thyroid cancer metastases, its role being considered supplementary to serum thyroglobulin (Tg) measurements and radioactive iodine (131I) whole-body scans (WBS). The present retrospective study was designed to elucidate the role of 99mTc-MIBI scans in localizing metastatic lesions by assessing sensitivity and specificity of the scan results obtained in a group of 68 thyroidectomized thyroid cancer patients. Presence or absence of thyroid cancer was judged with other diagnostic modes including serum Tg measurements, 131I WBS, bone scans, chest x-rays, computed tomography (CT), ultrasonography, histopathology, and evolution of disease during follow-up. All scans were read on lesion basis for detecting neck, lung, and bone metastases and also on region basis, namely head-neck, chest, and abdomen-pelvis-extremities (ab-p-ex) areas. The sensitivity of detection was 94.4% (17/18) for neck, 78.4% (40/51) for lung, and 92.8% (64/69) for skeletal lesions. Positive predictive value (PPV) and negative predictive value (NPV) were 96.3% (26/27) and 97.7% (43/44) for head-neck; 94.7% (71/75) and 50.0% (12/24) for chest; 100.0% (25/25) and 93.1% (54/58) for ab-p-ex regions, respectively. For all scan sites taken together, PPV and NPV were 96.1% (122/127) and 86.5% (109/126), respectively. In conclusion, the present study reveals that 99mTc-MIBI can be proposed as a first-line diagnostic agent for the follow-up protocol of thyroid cancer patients, although the ability to detect small lung metastases is somewhat limited.

Epigallocatechin gallate-induced histamine release in patients with green tea-induced asthma.

BACKGROUND AND OBJECTIVE: Epigallocatechin gallate is the causative agent of green tea-induced asthma. To determine whether an IgE-mediated mechanism plays a pathogenetic role in this disorder, we measured histamine release after in vitro exposure to epigallocatechin gallate. METHODS: Subjects included eight patients (four men and four women) with green tea-induced asthma, who had been diagnosed by skin test and inhalation challenge, and eight controls (four asthmatic subjects with no previous exposure tea dust and four healthy volunteers). Heparinized whole blood samples were taken and incubated with epigallocatechin gallate at various concentrations (final concentration range, 0.003 to 300 micrograms/mL) for 30 minutes at 37 degrees C. After centrifugation, histamine was measured in the cell-free supernatants by radioimmunoassay. Histamine release was expressed as a percentage of total histamine. A result higher than 10% was considered positive. RESULTS: In one of the tea-sensitive patients, epigallocatechin gallate did not cause histamine release. Five of the other seven patients (71%) demonstrated a positive, dose-dependent histamine release to epigallocatechin gallate. In asthmatic and normal controls, histamine release was not observed at any epigallocatechin gallate concentration. Furthermore, a significant correlation was noted between the maximum percentage histamine release and the threshold epigallocatechin gallate concentration for intradermal skin testing. CONCLUSION: These results indicate that an IgE-mediated response is the basis for green tea-induced asthma.

[Prophylactic effect of black tea extract as gargle against influenza].

We examined whether gargling with black tea prevents influenza infection. Tests were carried out during a five month period (October 1992 to March 1993). The control group that followed their normal daily routine, whereas the test group that gargled with 0.5 w/v% black tea extract twice daily (at 8 a.m. and 5 p.m.). Influenza viruses were isolated from influenza patients and an antigen analysis was carried out. As a result, two strains of influenza A viruses (H3N2) and ten strains of B virus were detected. An HI test was done using paired sera of the control group and the test group. The HI titers raised a four fold or greater in 48.8% (61/125) in the control group and 35.1% (35/134) in the test group. There was a significant difference (p < 0.05) between the control and test groups. These results indicate that black tea extract is effective as a prophylactic agent against influenza infection.

Development of a time-resolved fluoroimmunoassay (TR-FIA) for testosterone: measurement of serum testosterone concentrations after testosterone treatment in the rainbow trout (Oncorhynchus mykiss).

A sensitive time-resolved fluoroimmunoassay (TR-FIA) for testosterone was developed, and the assay system was used for measuring serum testosterone concentrations in rainbow trout. Testosterone-3-(O-carboxymethyl)oxime-bovine serum albumin (T-3-CMO-BSA) was immobilized by physical adsorption to the wells of microtiter plates. A competitive assay using two antibodies was performed among T-3-CMO-BSA in the solid-phase, unknown amounts of testosterone, testosterone antibodies, and europium labeled secondary antibodies, followed by measurements using a time-resolved fluorometer (DELFIA system). The TR-FIA had a sensitivity of 0.075 pg/50 microliters sample (1.5 pg/ml), and the range of the assay system was between 1.5 pg/ml and 25 ng/ml. The intra- and interassay coefficients of variation for the testosterone TR-FIA were satisfactorily low, and were between 1.62 and 6.38% and 2.96 and 8.29%, respectively. The assay system was applied to measure the serum testosterone concentrations after an injection of testosterone dissolved in saline, propyleneglycol, or coconut oil. Among the three solvents, the coconut oil group showed continuously high serum testosterone level. In contrast, the saline and propyleneglycol groups had maximum concentrations 24 hr after the injection, but their levels were significantly lower than that of the coconut oil group. The testosterone TR-FIA method is sensitive, repeatable, and is as accurate as conventional RIAs. It is very good for measuring serum testosterone concentrations.

Effect of 6-fluoro-8-methoxy quinolone (AM-1155) against chronic airway infection with Pseudomonas aeruginosa in a rat model.

We studied the effect of AM-1155, a newly developed quinolone, against chronic airway infection with Pseudomonas aeruginosa using a previously described rat model. AM-1155 (25 mg/kg) or ciprofloxacin (25 mg/kg) or saline (controls) were injected s.c. for 14 days (from day 4 to day 17) after the inoculation of agar beads containing P. aeruginosa. The number of viable cells of intrapulmonary P. aeruginosa, histological findings of the lungs and immunoglobulin levels of serum and bronchoalveolar lavage fluid were examined in rats 11 and 18 days after the treatment. The findings indicated that the number of viable cells of P. aeruginosa in lungs was significantly decreased in the AM-1155- or ciprofloxacin-treated group compared with the non-treated control group. Histological examination in the non-treated control group showed hyperplasia of bronchus-associated lymphoid tissue as well as cellular infiltration in airways, but not prominently in the AM-1155- or ciprofloxacin-treated group. The IgG and IgA levels in serum and bronchoalveolar lavage fluid were significantly lower in the AM-1155- and ciprofloxacin-treated groups than in the control group. These in-vivo effects of AM-1155 were comparable to those of ciprofloxacin. These findings suggest that treatment with AM-1155 and ciprofloxacin suppressed excessive immune responses, preventing progression of airway damage in the chronic infectious state.

Responses of monkeys with pollinosis to two major allergens of Japanese cedar pollen.

We examined the responses of two Japanese monkeys with pollinosis to two major allergens (Cry j 1 and Cry j 2) of Japanese cedar pollen. The two monkeys (A and B) had specific IgE antibodies to the allergens and showed a strong positive reaction to both of them in the intradermal test. In the histamine release test with peripheral blood mononuclear cells (PBMC), monkey A showed a typical pattern similar to that seen in human patients, while monkey B released a low level of histamine. The proliferative response of PBMC to both allergens in monkey A was weak, but was typical in monkey B. From clinical as well as immunological points of view, these monkeys may be a suitable animal model for Japanese cedar pollinosis in humans.

Myoglobin inhibits proliferation of cultured human proximal tubular (HK-2) cells.

Following nephrotoxic injury, renal repair is dependent on tubular regeneration. In the case of myoglobinuric acute renal failure (ARF), persistence of myoglobin within tubular cells, or sublethal injury sustained at the height of exposure to it, might retard this process. To test this hypothesis, a human proximal tubular cell line (HK-2) was cultured for 24 hours in the absence or presence of clinically relevant myoglobin concentrations (0.5, 1, 2, 4 mg/ml). Immediately following myoglobin removal, lethal cell injury (vital dye uptake), lipid peroxidation, and DNA damage (alkaline unwinding assay) were assessed. The extent of cell proliferation was estimated over the next four days by a tetrazolium based (MTT) assay and by determining total intracellular LDH. Myoglobin's effects on protein and DNA synthesis were also assessed (35S-methionine and bromodeoxyuridine incorporation, respectively). Myoglobin induced dose-dependent lipid peroxidation (malondialdehyde generation) and cell death (up to 80% vital dye uptake with the 4 mg/ml challenge). Although 1 mg/ml myoglobin caused no cell death, it induced nearly complete growth arrest. This lasted for approximately three days following myoglobin removal from the media. Neither of two control proteins (albumin; lysozyme) nor a second nephrotoxin (gentamicin; 1 mg/ml) reproduced this effect. The 1 mg/ml myoglobin challenge caused an 80 to 90% depression in protein and DNA synthesis. It also induced significant DNA damage, as assessed by the alkaline unwinding assay (P < 0.01). Iron chelation therapy (deferoxamine) mitigated myoglobin-induced cell killing. However, its addition following myoglobin loading worsened HK-2 outgrowth by exerting a direct anti-proliferative effect. These results indicate that: (1) sublethal myoglobin toxicity can induce transient proximal tubular cell growth arrest, potentially slowing recovery from ARF; (2) this effect correlates with, and could result from, heme-induced DNA damage and a blockade in DNA/protein synthesis; and (3) deferoxamine can inhibit proximal tubular cell proliferation. This possibility needs to be considered in designing clinical trials with DFO for myohemoglobinuric ARF.

Comprehensive treatment of advanced neuroblastoma involving autologous bone marrow transplant.

Encouraging results are reported with high-dose chemotherapy and total body irradiation followed by autologous bone marrow transplantation in the treatment of advanced neuroblastoma. However, relapse remains a significant problem. We used high-dose chemotherapy, surgery, intraoperative radiation and an autologous bone marrow transplant treated in vitro to remove tumor cells followed by 13-cis-retinoic acid to treat 36 children with advanced neuroblastoma. This comprehensive treatment appears to improve the survival rate of patients with advanced neuroblastoma, including those with N-myc amplification and bony involvement. The disease-free survival rate was 66% (95% confidence interval, 49-84%) at 3 years. All patients who received 13-cis-retinoic acid developed cheilitis, but no bone marrow depression occurred in these patients. Five patients developed hemolytic uremic syndrome (HUS) post-transplant. This may have been related to the procedure used for total body irradiation. Patients who had their kidneys shielded during this procedure did not develop this syndrome. Patients who received local irradiation at the primary site showed no evidence of relapse in this region, indicating that such therapy may help to prevent a relapse. These data suggest a high rate of 3 year disease-free survival with this treatment strategy. The nonrandomized nature of the study and use of multiple modalities precludes analysis of the specific contribution of each.

Calcineurin activation protects T cells from glucocorticoid-induced apoptosis.

In T cell hybridomas, TCR/CD3 complex-mediated stimulation induces apoptosis but inhibits that induced by glucocorticoids. A combination of ionomycin (IM), a calcium ionophore, and PMA, a protein kinase C activator, mimics the effects of the TCR/CD3-mediated stimulation. Glucocorticoid-induced apoptosis is, however, markedly inhibited by IM alone, and less markedly by PMA alone. The immunosuppressant FK506 canceled the inhibition by IM but not that by PMA. As calcineurin (CN) is one of the target molecules of FK506, we examined whether CN activation might have an anti-apoptotic effect. BOG8, a T cell hybridoma, was stably transfected with a mutant CN catalytic subunit with Ca2+/calmodulin-independent, constitutive but FK506-sensitive phosphatase activity. The transfectant clones were fairly resistant to glucocorticoid-induced death. Their resistance, however, was hardly affected by FK506 when added simultaneously with glucocorticoid, but was lost after a prolonged preincubation with FK506. In the parent BOG8 cells, FK506 failed to cancel the inhibitory effect of IM on glucocorticoid-induced death when the addition of FK506 was delayed for 1 h or more. These results suggest that CN activation is required for the resistance only as an early event. The transfectant clones produced IL-2 but failed to undergo apoptosis upon stimulation with PMA alone, whereas apoptosis was induced by a combination of IM and PMA. These results suggest that activation-induced cell death may require a higher level of CN activity than IL-2 production or may require another Ca(2+)-dependent pathway.

Autologous bone marrow transplantation in children with advanced neuroblastoma.

BACKGROUND: Encouraging results have been reported with high dose chemotherapy and total body radiation followed by bone marrow autotransplantation in children with advanced neuroblastoma; however, relapse remains a significant problem. METHODS: The authors treated 22 children with advanced neuroblastoma with high dose chemotherapy, surgery, intraoperative radiation, and a bone marrow autotransplant (treated in vitro to remove tumor cells) followed by 13-cis-retinoic acid. RESULTS: The 3-year relapse rate was 25% (95% confidence interval [CI], 6-44%). The 3-year disease free survival rate was 72% (95% CI, 52-92%). Toxicities included hemolytic uremic syndrome, herpes infection, and hepatic venoocclusive disease. CONCLUSION: These data suggest that this treatment strategy offers an increased rate of 3-year disease free survival. The nonrandomized nature of this study and its use of multiple modalities precludes the analysis of the specific contribution of each treatment component and comparison with conventional therapy.

Suppression of motor cortical excitability by electrical stimulation over the cerebellum in ataxia.

We studied the effect of electrical stimulation over the cerebellum on electromyographic responses evoked by magnetic stimulation over the cerebral motor cortex in 41 normal volunteers and 32 patients with ataxia due to various disorders. In all the normal subjects, stimulation over the cerebellum significantly reduced the size of electromyographic response in the first dorsal interosseous muscle evoked by magnetic cortical stimulation, when the cerebellar stimulus preceded the cortical stimulus by 5, 6, and 7 msec. This suppression was absent or reduced in ataxic patients who had atrophy of the cerebellar hemispheres as demonstrated by magnetic resonance imaging and in patients with dysfunction of the cerebellothalamocortical pathway who had lesions in the superior cerebellar peduncle or in the motor thalamus. In contrast, suppression was normal in ataxic patients who had pontine lesions that affected the pontocerebellar afferent pathway to the cerebellum. Results were also normal in patients without cerebellar ataxia, such as those with Parkinson's disease, sensory ataxia, and cerebrovascular disease without ataxia. We conclude that electrical stimulation activates cerebellar structures that suppress motor cortical excitability through a cerebellothalamocortical pathway and that the afferent systems to the cerebellum make no or little contribution to the effect. The technique described here would be useful for distinguishing ataxia due to lesions of cerebellar afferent pathway from other types of cerebellar ataxia.