Non-telecentric two-photon microscopy for 3D random access mesoscale imaging.

Diffraction-limited two-photon microscopy permits minimally invasive optical monitoring of neuronal activity. However, most conventional two-photon microscopes impose significant constraints on the size of the imaging field-of-view and the specific shape of the effective excitation volume, thus limiting the scope of biological questions that can be addressed and the information obtainable. Here, employing a non-telecentric optical design, we present a low-cost, easily implemented and flexible solution to address these limitations, offering a several-fold expanded three-dimensional field of view. Moreover, rapid laser-focus control via an electrically tunable lens allows near-simultaneous imaging of remote regions separated in three dimensions and permits the bending of imaging planes to follow natural curvatures in biological structures. Crucially, our core design is readily implemented (and reversed) within a matter of hours, making it highly suitable as a base platform for further development. We demonstrate the application of our system for imaging neuronal activity in a variety of examples in zebrafish, mice and fruit flies.

Effect of dietary ascorbate on lipogenesis and lipolysis activities in black sea bream, Acanthopagrus schlegelii.

To assess the effect of dietary ascorbate on lipid metabolism, 1-year black sea bream (Acanthopagrus schlegelii) were reared on a casein-based purified diet and an ascorbate fortified diet (1,100 mg of L: -ascorbyl-2- monophosphate-Mg/kg diet). The fortified ascorbate was effectively incorporated into the fish body and elevated muscle carnitine content. Fortifications of dietary ascorbate depressed activities of glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase as lipogenic enzymes in the hepatopancreas and intraperitoneal fat body. Starvation after feeding experiment activated carnitine palmitoyltransferase as a lipolysis enzyme in the hepatopancreas in both control and vitamin C(VC) groups, while the lipolysis activity was significantly higher in VC group. These results confirmed that dietary ascorbate depressed lipogenesis and activated lipolysis, i.e., influenced the lipid metabolism of black sea bream.

[Successful repair of blunt right atrial rupture; report of a case].

A 58-year-old male was compressed between 2 trucks during a traffic accident, and was brought to a local hospital with chest and back pain. Computed tomography showed pericardial effusion, and pericardiocentesis was performed. As cardiac rupture was suspected by the aspiration of blood, the patient was immediately transferred to our hospital. Pericaridocentesis was performed again with the introduction of a 7F sheath to release cardiac tamponade because the patient suddenly lost consciousness and showed respiratory arrest with shock just after arrival. After recovery from shock, the patient underwent median sternotomy. Surgery was performed without using cardiopulmonary bypass, and the rupture site in the right atrium was closed by direct surtures. The patient recovered without cardiac event and was discharged on the 46th postoperative day.

[Spontaneous pneumomediastinum].

We encountered 3 patients with spontaneous pneumomediastinum. The patients were a 14-year-old female, a 16-year-old male and an 18-year-old male. All 3 were previously healthy. They complained of chest pain or dyspnea. On admission, physical examination revealed subcutaneous emphysema. Chest computed tomography (CT) revealed pneumomediastinum and subcutaneous emphysema. All patients were treated with rest and all recovered in 7 to 9 days without any complication. Spontaneous pneumomediastinum is an uncommon, usually benign, self-limited disorder that usually occurs in young adults without any apparent precipitating factor or disease. Most patients require only conservative treatment.

Bactericidal activity of the nitroimidazopyran PA-824 in a murine model of tuberculosis.

The nitroimidazopyran PA-824 has potent in vitro activity against Mycobacterium tuberculosis, a narrow spectrum of activity limited primarily to the M. tuberculosis complex, and no demonstrable cross-resistance to a variety of antituberculosis drugs. In a series of experiments, we sequentially characterized the activity of PA-824 in an experimental murine model of tuberculosis. The minimal effective dose was 12.5 mg/kg of body weight/day. The minimal bactericidal dose (MBD) was 100 mg/kg/day. When PA-824 was used as monotherapy at the MBD, it exhibited promising bactericidal activity during the initial intensive phase of therapy that was similar to that of the equipotent dose of isoniazid in humans. In combination with isoniazid, PA-824 prevented the selection of isoniazid-resistant mutants. Perhaps more importantly, PA-824 also demonstrated potent activity during the continuation phase of therapy, during which it targeted bacilli that had persisted through an initial 2-month intensive phase of treatment with rifampin, isoniazid, and pyrazinamide. Together, these data strongly support further evaluation of PA-824 in combination with first- or second-line antituberculosis drugs to determine its potential contribution to the treatment of drug-susceptible or multidrug-resistant tuberculosis, respectively.

[Right delayed traumatic diaphragmatic hernia; report of a case].

An 80-year-old woman was admitted to our hospital with abnormal shadow on chest X-ray 8 years after a chest trauma during which multiple ribs on the right side were fractured causing hemothorax. A diagnosis of right delayed traumatic diaphragmatic hernia was based on the findings on plain X-ray and multislice computed tomography. We performed surgery via thoracic approach with thoracoscopic assist. The transverse colon, liver, and omentum were dislocated into the right thoracic cavity and hardly adhered to the lung. We successfully repaired the ruptured diaphragm. The postoperative course was uneventful and the patient was discharged on the 33rd postoperative day.

[Pulmonary thromboembolism after thoracoscopic pulmonary wedge resection; report of a case].

We encountered a case of acute pulmonary embolism after lung cancer surgery. The case was a 64-year-old female. She was admitted to our hospital with an abnormal shadow on chest X-ray. There was a past history of hypopituitarism medicated with steroids. Chest computed tomography (CT) demonstrated ground glass attenuation shadow measuring 10 mm in the left upper lobe. She underwent left thoracoscopic pulmonary wedge resection due to bronchioloalveolar cell carcinoma. Postoperatively, the patient suddenly complained of chest pain and dyspnea the day after surgery. Chest CT showed left and right pulmonary arterial thromboembolism. Thrombolytic and anticoagulation therapy with urokinase and heparin sodium were immediately started. Venography demonstrated thrombus located in the vein of the bilateral lower leg region. We inserted an inferior vena cava filter to prevent aggravation of pulmonary embolism. After 11 days, CT showed completed thrombolysis in the bilateral pulmonary artery. The patient was discharged on the 25th postoperative day, and has been followed with anticoagulation therapy.

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG.

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.

Anticipatory and reflexive neck muscle activities during voluntary rapid jaw opening and passive jaw depression in humans.

The characteristics of head movement during voluntary rapid jaw opening movement and passive jaw depression were investigated using accelerometers and electromyographs (EMG) on eight healthy examinees. Passive depressions were executed by means of load on the lower jaw, initiated either by examinees themselves or an experimenter. In the depression initiated by examinees, a head-extension movement that preceded the load to the lower jaw and anticipatory activities in the nuchal region of the trapezius muscle were observed. In the depression initiated by the experimenter, the anticipatory activities were not observed. In both of these cases, stretch reflexes were induced in the trapezius muscle. During voluntary rapid jaw opening, a head-extension movement nearly synchronized with the opening movement in the lower jaw acceleration, and dorsal-neck muscle activities accompanying the synchronized movement were observed. The peak timing of these neck-muscle activities preceded the latencies of the stretch-reflex activities observed in the jaw-depressed tasks, but no anticipatory activities were observed in the dorsal-neck muscles. We conclude that neither the anticipatory activities nor the reflex activities observed in the passive depressions have effects on the initial part of the dorsal-neck muscle activities, which are related to the head-extension synchronized with the voluntary lower-jaw opening movement.

Interaction of corticotropin-releasing factor and glucagon-like peptide-1 on behaviors in chicks.

Both corticortropin-releasing factor (CRF) and glucagon-like peptide-1 (GLP-1) inhibit food intake of chicks, but they also produce other behaviors. The present experiments were undertaken to clarify the interaction of CRF and GLP-1 regarding their anorectic actions as well as other behaviors. In Experiment 1, birds were injected intracerebroventricularly (i.c.v.), following a 3-h fast, with either saline, 0.1 microg of CRF, 0.1 microg of CRF+0.1 microg of GLP-1 or 0.1 microg of CRF+1 microg of GLP-1, and food intake was measured for 2 h. The injection of CRF decreased food intake, and CRF injected with GLP-1 suppressed food intake for up to 2 h. Birds were treated similarly in Experiment 2 in which the doses of CRF and GLP-1 were reversed. GLP-1 strongly suppressed food intake, and this effect was augmented by coadministration of CRF. In Experiment 3, the behaviors of chicks injected with saline, CRF (0.1 microg), GLP-1 (0.1 microg) or CRF (0.1 microg)+GLP-1 (0.1 microg) were monitored for the numbers of steps, vocalization and locomotion. Chicks were excited, moved more and vocalized loudly following injection of CRF, whereas an opposite response was seen with GLP-1. The behaviors were intermediate following the coinjection of the two peptides. In conclusion, CRF and GLP-1 interact in the chick brain, but the response depends on the behavior being measured.

Intracerebroventricular injection of exendin (5-39) increases food intake of layer-type chicks but not broiler chicks.

To clarify the involvement of endogenous glucagon-like peptide-1 (GLP-1) on feeding in chicks, we examined the central effect of GLP-1 antagonist, exendin (5-39) on food intake. Intracerebroventricular co-injection of exendin (5-39) with GLP-1 attenuated the anorexigenic effect of GLP-1 in layer-type chicks. Furthermore, exendin (5-39) enhanced food intake of layer-type chicks under ad libitum feeding. However, this effect was not observed in broiler chicks. Therefore, endogenous GLP-1 may be important in the regulation of feeding in layer-type chicks but not in broiler chicks.

Characterization of immortalized human chondrocytes originated from osteoarthritis cartilage.

Immortalized cloned human chondrocytes isolated from a normal (Ch-4, 8, N) and an osteoarthritis patient (Ch-8-OA) were established by introduction of recombinant SV40-adenovirus vector containing SV40 early gene. These cells exhibited continuous proliferative capacity in monolayer culture and showed chondrocytic characteristics in that they were positive for alkaline phosphatase and collagen type II. When cells were treated with IL-1alpha, the growth was inhibited. IL-1alpha induced the production of IL-6, GM-CSF and TNFalpha from immortalized chondrocytes. Significantly high amounts of cytokines including IL-6, GM-CSF and TNFalpha were produced from Ch-8-OA cells, even in the absence of IL-1alpha stimulation. Interestingly, TNFalpha, exogenously added into the culture, inhibited the growth of Ch-8-OA cells. Further studies are required to clarify the different mechanisms on chondrocytes originating from osteoarthritis cartilage underlying the biological reaction to various cytokines and the production of these cytokines as compared with chondrocytes from normal cartilages. However, the novel chondrocyte cell lines established in the present study may provide researchers with a useful model for studying the pathogenesis of osteoarthritis.

Suppression of food intake induced by corticotropin-releasing factor family in neonatal chicks.

Corticotropin-releasing factor (CRF), urocortin and urotensin I share amino acid sequences, and they inhibit food intake in mammals. CRF plays a potent role in decreasing food intake in avian species, but the effects of urocortin and urotensin I have not been investigated. Therefore, the effect of these three peptides on food intake in the neonatal chick was compared. In Experiment 1, birds were injected intracerebroventricularly (i.c.v.) with either 0, 0.01, 0.1 or 1 microg of urocortin following a 3-h fast, and food intake was measured for 2 h post-injection. Food intake was suppressed in a dose-dependent manner. Using a similar design in Experiment 2, the effect of urotensin I was investigated. Urotensin I appeared to suppress food intake in neonatal chicks more than urocortin did. In Experiment 3, the efficacy of CRF, urocortin and urotensin I was directly compared using one dose, 0.1 microg. The results indicated that the suppressive effect on food intake was strongest for CRF followed by urotensin I, then urocortin. These results suggest that the structure of receptors for the CRF family in chicks may be somewhat different than in mammals.

Generation of autologous tumor-specific T cell clones from a patient with adenosquamous carcinoma of the lung.

BACKGROUND: Adenosquamous carcinoma of the lung is not a common cancer, but its prognosis is worse than that of adenocarcinoma or squamous cell carcinoma. Therefore, new therapeutic strategies need to be developed to treat this type of lung cancer. Recently, vaccination using tumor antigens which are recognized by cytotoxic T lymphocytes (CTL) has been applied mainly to melanoma patients. We therefore attempted to establish T cell clones specific for autologous tumor cells (AT) from a patient with adenosquamous carcinoma in order to analyze the specific immune responses against AT. METHODS: A lung adenosquamous carcinoma cell line was established from a resected tumor obtained from a 72-year-old patient. Regional lymph node lymphocytes were stimulated weekly with CD80-transfected AT to induce CTL. The CTL activities were assessed by a standard (51)Cr release assay and by cytokine release. RESULTS: We succeeded in inducing an AT-specific CTL line. Using a limiting dilution method, eight T cell clones were established. AT-specific activity was observed in three CD8(+) T cell clones and one CD4(+) T cell clone out of the eight clones tested. Anti-HLA class I and anti-HLA-B/C mAbs inhibited IFN-gamma production from the AT-specific CD8(+) clones co-cultured with AT, thus indicating the restriction element to be HLA-B*5201 or HLA-Cw*1202. In contrast, the CD4(+) T cell clone recognized AT in an HLA class II-restricted manner. CONCLUSIONS: These results are the first demonstration of a successful induction of AT-specific T cell clones from a patient with lung adenosquamous carcinoma. It may therefore supply a possible way to apply specific immunotherapy to this type of lung cancer.

[Comparisons of factors affecting voiding disorders between patients with benign prostate hyperplasia and volunteers].

PURPOSE: The prostate size and motivation to visit clinics were investigated in patients with prostate hyperplasia. OBJECTS AND METHODS: One hundred ninety-five patients who had urinary symptoms and visited our outpatient clinic between September 1994 and October 1999 and 268 age-matched volunteers in Mitaka City who underwent a medical examination of the prostate in June 1997 were compared. International Prostate Symptom Score (IPSS), Quality Of Life Score (QOL score), residual urine volume, prostate volume and urinary flow rate were measured. RESULTS: The prostate volume of the volunteers was 20-25 cm3 irrespective of the age. The prostate size of the outpatients was larger than that of the volunteers for every age group. IPSS and QOL score were significantly higher in the outpatients than in the volunteers. Diurnal urinary frequency and sense of residual urine contributed to the discrimination index of the two groups more significantly than the other scores. There was a significant correlation between prostate volume and residual urine volume. The score of weak urinary steam was inversely and significantly correlated with peak urinary flow rate. CONCLUSIONS: There was no age-related enlargement of the prostate gland. The prostate gland was significantly larger in the patients than in the volunteers even in those in their fifties. Urinary frequency and sense of residual urine are important factors for men to seek and receive medical care.

Influence on force curve exerted by jaw tapping force.

The purpose of this study, which made use of visual biofeedback, was to determine how methods of regulating jaw tapping force differed depending on the strength of the tapping, using the force curve as an index. Nine healthy examinees were asked to make 30-35 jaw tapping movements, reproducing the defined target tapping force as accurately as possible. We measured the duration of the tooth contact phase, the time to peak force, the first time derivative of force (peak dF/dt), and the time to peak dF/dt. The results indicated that the duration of the tooth contact phase and the time to peak force increased with the target value (P < 0.01). As the target rose, the peak dF/dt increased significantly (P < 0.01), but the time to peak dF/dt was not significant (P=0.134). We found that the higher the target value, the greater the degree of dependency on feedback information. We also found that both the peak dF/dt and the time to peak dF/dt were determined for each examinee prior to movement.

Intracerebroventricular injection of agouti-related protein attenuates the anorexigenic effect of alpha-melanocyte stimulating hormone in neonatal chicks.

It is well known that alpha-melanocyte stimulating hormone (alpha-MSH) inhibits feeding via melanocortin receptor-4 (MC4R) in the mammalian brain. The anorexigenic effect of alpha-MSH is attenuated by agouti-related protein (AGRP), an antagonist for MC4R. Present studies were carried out to clarify whether human AGRP (86-132) antagonizes the anorexigenic effect of alpha-MSH in broiler chicks. Intracerebroventricular injection of AGRP attenuated the anorexigenic effect of alpha-MSH. Furthermore, AGRP stimulated food intake of layer-type chicks under an ad libitum feeding condition but not broiler chicks, suggesting that the orexigenic effect of AGRP is different between two breeds. These also imply that the extent of the anorexigenic effect of endogenous alpha-MSH is different among two breeds. This may be a part of the difference in food intake between two breeds.

Unfavorable prognosis of patients with non-small cell lung carcinoma associated with HLA-A2.

BACKGROUND: HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and, thus, play an important role in immune surveillance. Since 1970s there have been many reports of an increased frequency of one or more HLA haplotype in association with autoimmune disease, and malignancy. We studied types of HLA class I antigens in 204 resected non-small cell lung carcinoma (NSCLC) patients and also examined its correlation with clinicopathologic features and prognosis. METHOD: Serological typing for HLA class I antigens was performed using a microcytotoxicity test. The disease-free survival curves were calculated by the Kaplan-Meier method and then compared using the Logrank test. Multivariate analysis was carried out by Cox's proportional hazard method. RESULTS: The difference in disease-free survival time between the HLA-A2 present group and A2 absent group was significant (P = 0.040). The 3-year disease-free survival rate of all patients was 44% in HLA-A2 present group and 66% in A2 absent group. When a comparison was made within the group with stage I, expression of HLA-A2 was the only independent factor that affected survival time by multivariate analysis (P = 0.0457). CONCLUSIONS: Expression of HLA-A2 was considered as one of the unfavorable prognostic factors in NSCLC patients. Our results suggested expression of HLA-A2 in NSCLC patients was one of the mechanisms of escape from immune surveillance.

Eradication of murine brain tumors by direct inoculation of concentrated high titer-recombinant retrovirus harboring the herpes simplex virus thymidine kinase gene.

Implantation of retrovirus-producing cells within a tumor has been demonstrated to eliminate malignant brain tumors effectively in animal models. In our previous study, the implantation of high-titer retrovirus-producing fibroblasts into tumors resulted in highly efficient transduction in vivo. The transduced glioma cells migrated far from the implantation site, potentiating the induction of the remarkable bystander effect. It is also possible, however, that the implantation of murine fibroblast-derived virus-producing cells may induce an immune response in patients. In this study, we prepared retroviruses carrying the herpes simplex virus thymidine kinase (HTK) gene with titers of 1.4--2.5 x 10(11) colony-forming units (c.f.u.)/ml, and stereotactically inoculated only 3 microl of the HTK-bearing retroviruses into the brain tumors of mice. Following repetitive ganciclovir (GCV) intraperitoneal injection, effective killing of glioma cells in the mouse brain was observed. The transduction efficiency was nearly as high as that observed for the implantation of high-titer retrovirus-producing fibroblasts. Eighty percent of brain tumor-bearing mice were completely cured by our treatment protocol using concentrated HTK-harboring retroviruses. Our results suggest that repeated inoculations of high-titer retroviruses carrying the HTK gene followed by GCV treatment may be a promising strategy for the clinical treatment of malignant gliomas.