Photoperiodic control of TSH-beta expression in the mammalian pars tuberalis has different impacts on the induction and suppression of the hypothalamo-hypopysial gonadal axis.

Seasonal reproduction depends on photoperiod-regulated activation or suppression of the gonadal axis. Recent studies in quail have identified long-day induced TSH-beta expression in the pars tuberalis (PT) as a rapid trigger of gonadal activation. Thyroid-stimulating hormone (TSH) induces type 2 deiodinase (Dio2) in the ependymal cell layer (EC) of the infundibular recess to stimulate the gonadal axis. A similar mechanism is proposed in sheep and mice, but the experimental data on the temporal patterns of induction and suppression of TSH-beta and Dio2 expression are incomplete. In the present study, we examined the expression of TSH-beta and Dio2 in hamsters transferred from short- to long-day conditions for 9 days, and demonstrate the induction of TSH-beta and Dio2 on day 8 after transition. These data demonstrate the close relationship between TSH-beta and Dio2 expression in the inductive pathway. The temporal expression of TSH-beta and Dio2 in the suppressive pathway was also examined by s.c. melatonin injection, which mimics the transition from long to short days. Importantly, Dio2 expression in the EC is suppressed on day 1 after the onset of injection, whereas TSH-beta expression in the PT was not suppressed until day 10. These data suggest that regulated transcription of TSH-beta is involved in the induction of the gonadal axis in mammals, whereas the suppression of this axis is mediated by different mechanisms.

Fluorescent labeling of both GABAergic and glycinergic neurons in vesicular GABA transporter (VGAT)-venus transgenic mouse.

Inhibitory neurons play important roles in a number of brain functions. They are composed of GABAergic neurons and glycinergic neurons, and vesicular GABA transporter (VGAT) is specifically expressed in these neurons. Since the inhibitory neurons are scattered around in the CNS, it is difficult to identify these cells in living brain preparations. The glutamate decarboxylase (GAD) 67-GFP knock-in mouse has been widely used for the identification of GABAergic neurons, but their GAD67 expression was decreased compared to the wild-type mice. To overcome such a problem and to highlight the function and morphology of inhibitory neurons, we generated four lines of VGAT-Venus transgenic mice (lines #04, #29, #39 and #49) expressing Venus fluorescent protein under the control of mouse VGAT promoter. We found higher expression level of Venus transcripts and proteins as well as brighter fluorescent signal in line #39 mouse brains, compared to brains of other lines examined. By Western blots and spectrofluorometric measurements of forebrain, the line #39 mouse showed stronger GFP immunoreactivity and brighter fluorescent intensity than the GAD67-GFP knock-in mouse. In addition, Venus was present not only in somata, but also in neurites in the line #39 mouse by histological studies. In situ hybridization analysis showed that the expression pattern of Venus in the line #39 mouse was similar to that of endogenous VGAT. Double immunostaining analysis in line #39 mouse showed that Venus-expressing cells are primarily immunoreactive for GABA in cerebral cortex, hippocampus and cerebellar cortex and for GABA or glycine in dorsal cochlear nucleus. These results demonstrate that the VGAT-Venus line #39 mouse should be useful for studies on function and morphology of inhibitory neurons in the CNS.

A simple preparation method for mouse eosinophils and their responses to anti-allergic drugs.

OBJECTIVE AND DESIGN: A simple method for preparing mouse eosinophils was established, and the characteristics of the eosinophils were assessed including their responses to anti-allergic drugs. MATERIALS OR SUBJECTS: Mouse eosinophils were prepared from peritoneal exudate cells of BALB/c mice primed and boosted with antigen ovalbumin (OVA). METHODS: Surface phenotypes, migration activities and leukotriene C(4) (LTC(4)) production abilities of these eosinophils were examined. In addition, the effects of anti-allergic drugs, oxatomide and tranilast, on generation of LTC(4) from mouse eosinophils were examined. RESULTS: Eosinophils of mice boosted with OVA were phenotypically and functionally identical with human eosinophils. Around 1 x 10(7) eosinophils were obtained from mouse peritoneal exudate. It was found that these mouse eosinophils enabled to migrate in response to eotaxin as well as platelet-activating factor (PAF), and generated LTC(4) by IL-5 stimulation. Moreover, it was revealed that clinically used anti-allergic drugs inhibited LTC(4)-production dose-dependently. CONCLUSIONS: The present study provides a convenient method to obtain fully functional mouse eosinophils that are useful for drug screening and for evaluating implications of eosinophils in allergic responses.

Immediate maxillary reconstruction after malignant tumor extirpation.

AIMS: Immediate maxillary reconstruction after malignant tumor extirpation differs from other types of maxillary reconstruction. Our reconstruction algorithm is described in this article. METHODS: One hundred ninety-four patients who had undergone maxillectomy for malignant tumors were reviewed, and maxillectomy defects were classified with the method of Cordeiro and Santamaria. RESULTS: Mean total blood loss was 848 ml, and 71 patients died within 2 years after surgery. For type IIIa defects of the orbital floor, titanium mesh or vascularized bone or cartilage was used for reconstruction, but the rate of postoperative complications did not differ between titanium and autografts. Therefore, to reconstruct orbital floor defects we have recently used only titanium mesh. For type I or II defects, we use autografts for only selected cases. CONCLUSIONS: We strive to perform less-invasive reconstructive surgery after resection for maxillary malignancy.

Increased susceptibility to airway responses in CD40-deficient mice.

The interaction between CD40 and its ligand (CD154) is crucial for IL-12 production and effective humoral immunity such as IgE production. Although the interaction seems to play a crucial role in asthmatic inflammation, previous studies investigating the role of the CD40 and CD154 interaction in experimental animal models of asthma are complicated due to multistep reactions in developing asthma. Here, in order to investigate the role of CD40 in the effector phase in the development of airway responses, we used CD40-deficient mice backcrossed with mice transgenic for an ovalbumin (OVA)-specific TCR (TCRtg). Using intranasal OVA administration followed by aerosol inhalation of OVA, greater airway hyperreactivity and eosinophilia in bronchoalveolar lavage fluid (BALF) were observed in CD40-deficient mice backcrossed with TCRtg mice (CD40-/-/ TCRtg mice), compared with control littermates (CD40+/+/ TCRtg mice). CD4+ helper T cell subset analysis of lung draining lymph nodes revealed that the Th1 component was significantly decreased in CD40-/-/ TCRtg mice. Airway hyperreactivity and airway eosinophilia significantly correlated with the predomination of Th2 cells. Cytokine measurements in BALF also showed decreased IL-12 and the predominance of Th2 cells in CD40-/-/ TCRtg mice. These results suggest that CD40 may play a protective role in developing asthma in the phase after establishing specific memory T cells through the regulation of the balance between Th1 and Th2 cells presumably via induction of IL-12.

Characterization of the portal signal in a nonsteady hyperglycemic state in conscious dogs.

To characterize the "portal signal" in a nonsteady hyperglycemic state, the kinetic relationship between net hepatic glucose balance (NHGB) and either hepatic glucose load (HGL) or plasma insulin level was determined during glucose infusion using a catheter technique in 36 conscious dogs. Glucose was infused intraportally (Po group) and peripherally (Pe group) at 39, 56, and 83 micromol x kg(-1) x min(-1) over 2 h. There was a linear relationship between mean NHGB and either mean HGL or plasma insulin levels at each rate in either delivery (HGL: Po r = 0.99, Pe r = 0.95; insulin: Po r = 99, Pe r = 0.79). The threshold levels for net hepatic glucose uptake were 3.8 and 11.7 mmol/l for plasma glucose and 65 and 392 pmol/l for plasma insulin, respectively. The slope of the regression line against the abscissa was four times larger in portal than in peripheral delivery (HGL: Po 0.20 vs. Pe 0.05, P < 0.05; insulin: Po 0.19 vs. Pe 0.04, P < 0.05). These results suggest that the portal signal overrules the threshold of glucose for hepatic uptake by increasing hepatic extraction rate in a nonsteady hyperglycemic state.

Sentinel node biopsy for breast cancer patients in Japan.

Sentinel node biopsy may become a standard procedure to detect lymph node metastases in early breast cancer. Numerous studies have confirmed and demonstrated the reliability of the hypothesis of sentinel node biopsy, with a high identification rate and overall accuracy connected with dye-guided and radio-guided sentinel node biopsy. To assess the benefit of sentinel node biopsy, randomized clinical trials are underway in Western countries comparing sentinel node biopsy with conventional axillary lymph node dissection. In Japan, feasibility studies on sentinel node biopsy started in the mid-1990s. The dye and radiopharmaceuticals associated with sentinel node biopsy commonly used in Western countries are not available in Japan. Japanese investigators have attempted to perform sentinel node biopsy using other dyes and radiopharmaceuticals. The results from feasibility studies have been similar to those reported previously. In conclusion, sentinel node biopsy for breast cancer patients is successful in Japan. The current status and the problems are discussed.

Comparison of insulinotrophic actions of nateglinide with glibenclamide dissociated from absorption in conscious dogs.

Nateglinide is more rapidly absorbed than glibenclamide. Therefore, the different absorption kinetics of both drugs were eliminated by intraportal administration in conscious fasted dogs. The plasma insulin profiles were compared under similar kinetic changes in plasma drug concentrations. After a priming dose of nateglinide (1 mg/kg. 5 min) or glibenclamide (40 microg/kg. 5 min), plasma drug concentrations reached a peak at 4 minutes (nateglinide, 80 +/- 5 micromol/L, n = 6 and glibenclamide, 263 +/- 60 nmol/L, n = 6) followed by a sustained level at approximately 30% of the peak concentration at 30 minutes. Nateglinide led to a rapid and constant reduction in arterial glucose of approximately 30% basal, while glibenclamide promoted a gradual decrease to approximately 50% basal at 120 minutes. An increase in plasma insulin level by nateglinide of 4 times basal (218 +/- 58 pmol/L v 47 +/- 3 pmol/L, P <.05, n = 6) occurred at 6 to 10 minutes followed by sustained release of 1.4 times basal (67 +/- 15 pmol/L, n = 6). The insulin surge was more than doubled (484 +/- 209 pmol/L, n = 6) under a euglycemic clamp. Insulin release by glibenclamide increased gradually reaching 10-fold basal (449 +/- 166 pmol/L, n = 6) at 60 minutes. This was not enhanced during a euglycemic clamp. Lowering the primed doses of nateglinide resulted in a diminished peak plasma insulin concentration. In contrast, glibenclamide caused only a slower increase, but eventually reaching a similar peak. By increasing the continuous infusion of nateglinide, the sustained insulin release was not altered. Glibenclamide, but not nateglinide, evoked prompt and sustained insulin release in the continuing presence of the other. These results are consistent with the concept that nateglinide produces a quick, but very short-lived, interaction with sulfonylurea (SU)-receptors on plasma membrane by free access of the drug from the cell exterior. In contrast, glibenclamide promotes a slow and longer interaction with the receptor by distribution of the drug into the cell inferior. We conclude, therefore, that not only the different kinetics of gastrointestinal (GI) absorption, but also the inherent difference in the interaction with beta cells is attributed to the different insulin release characteristics between nateglinide and glibenclamide in vivo.

Forced myofiber regeneration promotes dystrophin gene transfer and improved muscle function despite advanced disease in old dystrophic mice.

Duchenne muscular dystrophy (DMD) is caused by defects in the dystrophin gene. In young dystrophic mdx mice, immature regenerating myofibers represent the principal substrate for adenovirus vector (AdV)-mediated dystrophin gene transfer. However, in DMD patients immature regenerating myofibers are generally sparse. Such a situation also exists in old mdx mice, which may represent a more realistic model. Therefore, here we have used old mdx mice (of 14- to 17 months of age) to test the hypothesis that one-time administration of a myonecrotic agent can transiently re-establish a population of immature myofibers susceptible to AdV-mediated dystrophin gene transfer. This strategy led to upregulation of the coxsackie/adenovirus attachment receptor by means of induction of regenerating myofibers, significantly augmented AdV-mediated dystrophin gene expression, and enhanced force-generating capacity. In addition, it led to an increased resistance to contraction-induced injury compared with untreated controls. The latter protective effect was positively correlated with the number of dystrophin-expressing myofibers (r=0.83, P<0.05). Accordingly, the risk:benefit ratio associated with the sequential use of forced myofiber regeneration and AdV-mediated dystrophin gene transfer was favorable in old mdx mice despite advanced disease. These findings have implications for the potential applicability of AdV-mediated gene therapy to DMD and other muscle diseases in which immature regenerating myofibers are lacking.

[Surgical treatment of carcinoma of the hypopharynx and cervical esophagus].

This paper discusses several recent advances in surgical methods for treatment of cancer of the hypopharynx and cervical esophagus. The standard surgical technique for the primary lesion is laryngo-pharyngo-esophagectomy in which the larynx is usually resected to prevent postoperative aspiration even if the cancer does not directly involve the larynx. Another common technique is total laryngectomy plus partial resection of the hypopharynx, where a very limited lesion in the unilateral pyriform sinus is resected with the surrounding hypopharyngeal mucosa and larynx. In this case, the defect in the hypopharyngeal mucosa is primarily sutured or reconstructed with a graft based on its size. Experience has demonstrated that the larynx can be preserved without any postoperative aspiration if it is not involved by cancer and surgeons design the lines of resection and the postoperative shape of the reconstructed area to prevent aspiration. It has also been demonstrated that even if a part of the larynx is involved and must be resected, the remaining portion of the larynx can sometimes be preserved without any distinct aspiration. There are two common surgical techniques for neck lymph nodes. Radical neck dissection is the classic one, in which the lymphatic tissues together with the surrounding structures, including the sternocleidomastoid muscle, internal jugular vein, and accessory nerve are resected. Conservative neck dissection resects the lymphatic tissues only and preserves other structures. Currently, the standard surgical technique is conservative neck dissection. Radical neck dissection is rarely performed now because its morbidity is much higher and its superiority in treatment results has not been established.

Image analysis of microvessel surface area predicts radiosensitivity in early-stage laryngeal carcinoma treated with radiotherapy.

PURPOSE: The tissue oxygenation level, which is theoretically governed by distance from blood vessels, is one of the most important modulators of the radiosensitivity of carcinoma. A computed image analysis system for the detection of tissue oxygenation was developed to establish a method of predicting radiosensitivity in early-stage laryngeal carcinoma treated by curative radiotherapy. EXPERIMENTAL DESIGN: Microvessel structures labeled with CD31 antigen were investigated in 55 patients undergoing curative radiotherapy for T1 and T2 laryngeal carcinoma. We calculated (a) microvessel density [(MVD) vessels/field] under a microscope; (b) the ratio of the total microvessel number (TN):tumor area (TA) [TN:TA; vessels/mm2]; (c) the ratio of the total microvessel perimeter (TP):TA (TP:TA; mm/mm2); and (d) the ratio of tumor tissue area >150 microm from microvessels (hypoxic ratio; %) as parameters of tissue oxygenation in each whole biopsy specimen by using an image analyzer. We compared each of these factors with radiosensitivity. RESULTS: Mann-Whitney's U test revealed that tumors with a high MVD (median, 42 vessels/field), high TN:TA ratio (median=40.9 vessels/mm2), high TP:TA ratio (median, 2.92 mm/mm2), and low hypoxic ratio (median, 30.3%) had significantly greater radiosensitivity than tumors with a low MVD, low TN:TA ratio, low TP:TA ratio or high hypoxic ratio (P = 0.002, P = 0.0004, P < 0.0001, and P = 0.004, respectively). CONCLUSIONS: Prediction of radiosensitivity on the basis of the TP:TA ratio can be used as an efficient means of avoiding ineffective radiation, complications after salvage surgery, and prolonged hospital stays.

Deep circumflex iliac perforator flap with iliac crest for mandibular reconstruction.

The deep circumflex iliac myocutaneous perforator (DCIP) flap with iliac crest was used to reconstruct oromandibular defects in 10 patients. In seven of the patients a dominant perforator was found preoperatively using a Doppler flowmeter; in five of these seven patients a DCIP flap was successfully transferred. In two of the seven patients the dominant perforators were too narrow: one patient underwent a standard osteocutaneous flap transfer and one patient underwent a second flap transfer. In three patients no dominant perforator was found before or during surgery. The freedom of the DCIP flap from the harvested iliac crest facilitates correct positioning. However, to ensure that the DCIP flap can be safely elevated, the presence of perforators must be confirmed preoperatively. Even when a perforator has been identified, complicated dissection may be necessary. We stress the importance of a thorough knowledge of the anatomy of second flaps and of obtaining informed consent to use them.

Influence of postsurgical residual tumor volume on local control in radiotherapy for maxillary sinus cancer.

BACKGROUND: The aim was to study the influence of postsurgical gross residual tumor volume on local control of maxillary sinus cancer treated with radiotherapy combined with debulking surgery. METHODS: Forty-three patients who underwent combined surgery and radiotherapy (50-72 Gy, median 60 Gy) for squamous cell carcinoma of the maxillary sinus were reviewed. Gross residual tumor volume (GRTV) after surgery was measured on computed tomograms obtained during the radiotherapy planning. Patients were classified according to GRTV as follows: group AA, GRTV = 0 (microscopic residual, n = 2); group A, GRTV < 10 cm3 (n = 24); group B, 10-40 cm3 (n = 9); and group C, > or = 40 cm3 (n = 8). The relationship between local control and GRTV was analyzed using univariate and multivariate analysis. RESULTS: The 2-year local control rate for all patients was 62%. The differences in local control rates between groups AA, A and B were not significant (P > 0.05), but the rate was significantly lower in group C than in the other groups (69% at 2 years vs 31% at 1 year, P < 0.001). Multivariate analysis showed that GRTV (P = 0.002) and histological differentiation (poorly differentiated histology was favorable, P = 0.035) were independent prognostic factors and that intra-arterial chemotherapy and administered total dose were not. Local control in groups A and B significantly depended on the total dose of radiotherapy, with 2-year control rates of patients receiving 50 Gy (n = 6) and > or = 60 Gy (n = 27) of 17% vs 79%, respectively (P < 0.001). CONCLUSIONS: Our data suggest that adequate, not complete, debulking associated with a total radiotherapy dose of > or = 60 Gy can provide satisfactory local control for patients with squamous cell carcinoma of the maxillary sinus.