Tumour-infiltrating CD8 to FOXP3 lymphocyte ratio in predicting treatment responses to neoadjuvant chemotherapy of aggressive breast cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) can be used to monitor the immune response, and are important in predicting treatment responses and outcomes for various types of cancer. Recently, specific TIL subsets have been reported to be clinically useful in predicting treatment responses. The CD8+/FOXP3+ TIL ratio (CFR) may be a more sensitive indicator for monitoring immune function. This study investigated the clinical significance and value of CFR as a biomarker to predict treatment responses to neoadjuvant chemotherapy for breast cancer. METHODS: Patients with resectable early-stage breast cancer treated with neoadjuvant chemotherapy at Osaka City University Hospital, Japan, between 2007 and 2013 were included. Oestrogen receptor, progesterone receptor, human epidermal growth factor receptor (HER) 2, Ki-67, CD8 and FOXP3 status were assessed by immunohistochemistry, and correlated with pathological complete response (pCR). RESULTS: A total of 177 patients were included, of whom 90 had a high CFR and 87 a low CFR. Triple-negative breast cancer (TNBC) was more common in the high-CFR group than in the low-CFR group (46 versus 23 per cent; P = 0·002), as was HER2-enriched breast cancer (HER2BC) (27 versus 14 per cent; P = 0·033). Among these patients, the pCR rate was significantly higher in the high-CFR group than in the low-CFR group (TNBC: P = 0·022; HER2BC: P < 0·001). In multivariable analysis high-CFR status was an independent predictor of a favourable prognosis: hazard ratio 0·24 (95 per cent c.i. 0·05 to 0·72; P = 0·015) for TNBC and 0·10 (0·10 to 0·90; P = 0·041) for HER2BC. CONCLUSION: The CFR may be a useful biomarker to predict treatment response to neoadjuvant therapy in aggressive breast cancer subtypes, such as TNBC and HER2BC.

c-Kit expression as a prognostic molecular marker in patients with basal-like breast cancer.

BACKGROUND: As patients with basal-like breast cancer (BLBC) have a poor prognosis and there is no specifically tailored therapy, molecular biological characterization of BLBC is necessary. c-Kit is a transmembrane receptor tyrosine kinase known to play important roles in various solid cancers. This study classified BLBCs from patients with breast carcinoma, and addressed the significance of c-Kit expression in these tumours. METHODS: Primary breast tumours were stained with antibodies against oestrogen receptor, progesterone receptor, human epidermal growth factor receptor (HER) 2, epidermal growth factor receptor (EGFR), cytokeratin 5/6 and c-Kit. The association between c-Kit, BLBC and survival was analysed. RESULTS: A total of 667 patients with breast cancer were followed up for a median of 39 (range 6-72) months. Some 190 tumours (28·5 per cent) were classified as triple-negative for breast cancer (negative for oestrogen receptor, progesterone receptor and HER2) and 149 (78·4 per cent) had characteristics of BLBC (positive for cytokeratin 5/6 and/or EGFR). c-Kit expression was detected in 111 (16·6 per cent) of 667 tumours. c-Kit-positive tumours were more commonly found among patients with BLBC (42 of 149, 28·2 per cent; P < 0·001) and in patients with nodal metastasis (47 of 216, 21·8 per cent; P = 0·014) than in those without. In patients with BLBC, the prognosis was significantly worse in those with c-Kit expression (P < 0·001). Multivariable logistic regression analysis revealed c-Kit as an independent negative prognostic factor for cancer-specific survival in patients with BLBC (hazard ratio 2·29, 95 per cent confidence interval 1·11 to 4·72). CONCLUSION: c-Kit might be a prognostic marker and possible molecular target for therapy in patients with BLBC.

Epidermal growth factor and transforming growth factor alpha induce ascitic fluid in mice.

Recent studies have suggested that pleural or peritoneal effusion associated with metastatic tumors is induced by some mediators produced by the tumor cells. We studied the ability of well-characterized peptide growth factors to produce ascites in mice. Peritoneal administration of epidermal growth factor (EGF, 10 to 40 micrograms/mouse/wk) or transforming growth factor alpha (TGF-alpha, 10 to 40 micrograms/mouse/wk) via osmotic minipumps resulted in formation of bloody ascites. The amount of ascites produced was dependent on the dose of growth factors. Vehicle alone or insulin-like growth factor I (40 micrograms/mouse/wk) was without effect. Indomethacin, a blocker of prostaglandin synthesis, significantly reduced the ascites accumulation induced by EGF, suggesting that prostaglandins are involved in ascites formation induced by EGF. Dexamethasone was also effective in attenuating the effect of EGF. Thus, it is possible that peritoneal effusion associated with disseminated tumors is, at least in part, due to EGF-like materials (most likely TGF-alpha) produced by tumor cells. The mechanism by which these peptides induce bloody ascites is not known for certain, but it may be due to the reported activity for neovascularization or increased vascular permeability.

Insulin-like growth factor I and transforming growth factor alpha as autocrine growth factors in human pancreatic cancer cell growth.

The roles of insulin-like growth factor I (IGF-I) and transforming growth factor alpha (TGF-alpha) as autocrine factors in the proliferation of MIA-PaCa 2 cells (human pancreatic cancer cells, PC cells) were investigated. Furthermore, the mechanism(s) of inhibition of PC cell growth by a phorbol ester in relation to these two kinds of growth factor was also studied. PC cells grew autonomously when Dulbecco's modified essential medium supplemented with 4% fetal calf serum was changed to serum-free medium (0.3% bovine serum albumin-Dulbecco's modified essential medium). In addition, serum-free conditioned medium from PC cells dialyzed against fresh Dulbecco's modified essential medium had a stimulatory action on the growth of the same kind of cells when compared with that induced by nonconditioned medium. These observations suggest that a factor(s) produced and released by PC cells stimulates their own growth. Analysis of conditioned medium from PC cells revealed the presence of immunoreactive (IR)-IGF-I and IR-TGF-alpha. The molecular size of IR-IGF-I was similar to that of authentic IGF-I. On the other hand, IR-TGF-alpha was present as multiple forms when analyzed using gel chromatography. Authentic IGF-I and TGF-alpha added to culture medium stimulated PC cell growth by 1.45- and 1.5-fold above control value, respectively. A monoclonal antibody to IGF-I receptor was able to inhibit PC cell growth. PC cell proliferation was markedly inhibited by 12-O-tetradecanoyl-13-acetate (greater than 0.16 nm), whereas cell growth of human fibroblasts was stimulated by it. 12-O-Tetradecanoyl-phorbol-13-acetate also reduced the binding of 125I-TGF-alpha, but not 125I-IGF-I, to PC cells. Decrease in TGF-alpha binding was mainly due to the reduced affinity of receptors to the ligand. These results suggest that IGF-I and TGF-alpha are involved in PC cell proliferation as autocrine factors. Further, the inhibition of PC cell growth by phorbol ester could be, at least partly, due to the decreased binding of TGF-alpha to the cells.

Tumor angiogenesis as a predictor of recurrence in gastric carcinoma.

PURPOSE: We investigated the correlation between tumor angiogenic activity and progression of gastric carcinoma using immunohistochemical staining with antifactor VIII-related antigen (F-VIII RAg) antibody. MATERIALS AND METHODS: One hundred twenty-four specimens resected from patients with gastric carcinoma were investigated by staining with a monoclonal antibody against F-VIII RAg. Correlations between the microvessel count (the mean number of microvessels in the five areas of highest vascular density at 200 times magnification), various clinicopathologic factors, and prognosis were studied. RESULTS: The microvessel count increased with histologic stage. The microvessel count was significantly higher in patients with lymph node metastases than in those without such metastases. Moreover, in patients with a high microvessel count (> or = 16), prognosis was significantly poorer than in those with low count (< 16). Multivariate analysis indicated that the microvessel count is an independent prognostic factor in patients with gastric cancer. According to the mode of recurrence, the frequency of hepatic metastases was significantly increased in patients with a high count. CONCLUSION: Microvessel count may be a good prognostic indicator and may be useful as a predictor for the mode of recurrence in patients with gastric carcinoma.

Effects of N-methyl-D-aspartate glutamate receptor antagonists on oscillatory signal propagation in the guinea-pig accessory olfactory bulb slice: characterization by optical, field potential and patch clamp recordings.

To characterize the role of N-methyl-d-aspartate glutamate receptors in oscillations induced by a single electrical stimulation of the vomeronasal nerve layer, optical, field potential and patch clamp recordings were carried out in guinea-pig accessory olfactory bulb slice preparations. Bath application of the N-methyl-D-aspartate receptor antagonists, 2-amino-5-phosphonovaleric acid or MK-801, produced an increase in frequency of oscillating waves (oscillation) in external plexiform and mitral cell layers. The removal of Mg2+ from perfusate abolished oscillations, while subsequent application of 2-amino-5-phosphonovaleric acid or MK-801 restored oscillations. Vomeronasal nerve layer-evoked postsynaptic currents were analyzed by whole-cell clamp recordings from mitral and granule cells. A long-lasting excitatory postsynaptic current and periodic inhibitory postsynaptic currents, which were superimposed on the long excitatory postsynaptic current, were observed in mitral cells. The frequency of the periodic inhibitory postsynaptic currents correlated with the frequency of oscillations observed in the optical and field potential recordings. Furthermore, periodic inhibitory postsynaptic currents were blocked by puff application of bicuculline to the external plexiform layer/mitral cell layer, where mitral cells make dendrodendritic synapses with granule cells. In addition, puff application of the non-N-methyl-D-aspartate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, to the external plexiform layer/mitral cell layer suppressed an early phase of periodic inhibitory postsynaptic currents (membrane oscillation), whereas 2-amino-5-phosphonovaleric acid suppressed the late phase of periodic inhibitory postsynaptic currents. These data indicate that periodic excitatory postsynaptic currents of granule cells induce relevantly periodic inhibitory postsynaptic currents in mitral cells via dendrodendritic synapses and suggest that feedback inhibition regulates generation of oscillation via activation of non-N-methyl-d-aspartate glutamate receptors and gradual attenuation of oscillation via activation of N-methyl-D-aspartate receptors on granule cells.

Developmental changes in oscillatory and slow responses of the rat accessory olfactory bulb.

Field potential, patch-clamp and optical recordings were performed in accessory olfactory bulb slices of postnatal rats following single electrical stimulation of the vomeronasal nerve layer. On the basis of differences in the components of the field potential, postnatal days were divided into three periods: immature (until postnatal day 11), transitional (postnatal days P12-17) and mature periods (after postnatal day 18). During the immature period, vomeronasal nerve layer stimulation provoked a characteristic damped oscillatory field potential, and the field potential recorded in the glomerular layer consisted of a compound action potential followed by several periodic negative peaks superimposed on slow components. Reduction in [Mg2+] enhanced slow components but did not affect oscillation, whereas an NMDA receptor antagonist, D-2-amino-5-phosphonovalerate, depressed slow components but did not affect the oscillation. During the mature period, slow components and the periodic waves (oscillation) disappeared. The time course of the field potential was similar to that in adults, suggesting that the accessory olfactory bulb reached electrophysiologically maturity at postnatal day 18. A non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, inhibited vomeronasal nerve layer-induced responses, while D-2-amino-5-phosphonovalerate had no effect, suggesting that NMDA and non-NMDA receptors are active in immature tissues, whereas non-NMDA receptors predominated in mature tissue. Results from whole-cell patch recordings in mitral and granule cells yielded results consistent with those from field potential and optical recordings. Further, a gradual decrease in number and frequency of oscillating waves was observed until postnatal day 17. Analyses of the depth profile of field potentials and current source density in immature tissue suggested that the oscillation and slow components originated in the glomerular layer but not in the external plexiform/mitral cell layer. Further, a new type of oscillation, which was independent of the reciprocal dendrodendritic synapses between mitral and granule cells, was detected. These data indicate that the lack of oscillatory suppression by immature NMDA receptors may play a critical role in the dynamic alteration of bulbar conditions.

Odor-concentration coding in the guinea-pig piriform cortex.

By optical imaging of intrinsic signals, we demonstrated a possible code for odor concentration in the anterior piriform cortex of the guinea-pig. Odor-induced cortical activation, which primarily originated in layer II, appeared in a narrow band beneath the rhinal sulcus over the lateral olfactory tract, corresponding to the dorsal part of the anterior piriform cortex. Lower concentrations activated the rostral region of the band, whereas higher ones generated caudally spreading activation, and the site at which neural activation reached its maximum extent depended upon odor concentration. Different odors with low concentrations generated distinct but somewhat overlapping patterns in the rostral region of the band; the limited extent of cortical activity may be one focal domain for each odor. It was hard to judge, however, that odor-specific domains appeared in the anterior piriform cortex, because the strong stimuli induced largely overlapping patterns. Furthermore, the total area activated increased in proportion to concentrations raised to a power of 0.5-0.9. Importantly, these imaging results were confirmed with unit recordings which indicated a rostro-caudal gradient in odor-sensitivity among cortical neurons. Our results suggest that the dorsal part of the anterior piriform cortex may be associated with odor concentration. Therefore, in addition to recruitment of more olfactory sensory cells and glomeruli in response to stronger stimuli, a rostro-caudal gradient in axonal projections from mitral/tufted cells and/or in association fibers may play an important role in odor-concentration coding in the anterior piriform cortex.

Efficacy of Helicobacter pylori eradication on the chronic mucosal inflammation of the remnant stomach after distal gastrectomy for early gastric cancer.

Although eradication of Helicobacter pylori (Hp) after early gastric carcinoma has been recommended, very limited studies have been reported and the method differs from standard therapy. Here, we attempted the eradication of Hp in the remnant stomach after surgery for primary gastric cancer with the standardized method. We examined efficacy and the safeness of the treatment. Thirty-three H. pylori-positive patients after distal gastrectomy were treated with proton pump inhibitor (PPI)-based triple therapies. After eradication, endoscopic and histological changes were classified on the basis of the Updated Sydney System. The eradication rate in the remnant stomach was 90.9% (30 out of 33 cases) after triple therapy. Temporal minor side effects were notified in 3 cases. After eradication, the remnant stomach showed significant decreases in inflammation- and activity-scores. Moreover, significant improvement in glandular atrophy to normal mucosa was found. In conclusion, PPI-based standard therapy is just as effective for Hp eradication in the remnant stomach than it is in the non-operative stomach. Eradication therapy could be performed safely and resulted in a significant improvement in inflammation and atrophy of the mucosal layer in the remnant stomach after early gastric cancer surgery.

Effects of retinoids on iodine metabolism, thyroid peroxidase gene expression, and deoxyribonucleic acid synthesis in porcine thyroid cells in culture.

Effects of retinoids on DNA synthesis, iodine metabolism, and thyroid peroxidase messenger RNA levels were studied in cultured porcine thyroid cells. Retinol (10(-8)-10(-5) M) alone did not affect DNA synthesis but potentiated that induced by epidermal growth factor or insulin-like growth factor-I without changes in the number or affinity of receptors for the growth factors, suggesting that retinol stimulates postreceptor events responsible for DNA synthesis. Retinol was an inhibitor of TSH-stimulated iodine metabolism. Iodide uptake and release of organified iodine stimulated by TSH or forskolin were inhibited dose dependently by treatment with retinol. The inhibition was detected at 10(-8) M and was approximately 50% at 10(-6) M. The potency of retinoic acid was comparable to that of retinol. The inhibitory effect of retinol was detected after treatments of thyroid cells for 24 h, and the maximal effect occurred after 48 h incubation. The cAMP accumulation in cultures treated with TSH plus retinol was lower than that of control cultures treated with TSH alone. However, iodide uptake stimulated by 8-bromo-cAMP was also inhibited by retinoids. Retinol reduced TSH- or 8-bromo-cAMP-stimulated gene expression of thyroid peroxidase. Thus, the data suggest that retinoids inhibit TSH-stimulated iodine metabolism by reducing cAMP accumulation and also by acting on the steps subsequent to cAMP production.

Increased concentration of vascular endothelial growth factor/vascular permeability factor in cyst fluid of enlarging and recurrent thyroid nodules.

Human thyrocytes produce vascular endothelial growth factor (VEGF), a potent angiogenic factor, also known as vascular permeability factor (VPF), which increases vascular permeability. Based on the assumption that VEGF/VPF is involved in fluid accumulation in thyroid cysts, we determined the VEGF/VPF concentration in cyst fluids of thyroid nodules from 79 patients. VEGF/VPF was found to be abundantly present in the cyst fluids (0.02-183 ng/mL). There was no significant difference of VEGF/VPF concentration in the cyst fluid obtained from thyroid adenoma or from adenomotous goiter with cystic degeneration. Immunoreactive VEGF/VPF in cyst fluid was eluted mainly at 45 kDa, and stimulated endothelial cell proliferation, which was partially blocked by anti-VEGF/VPF antibody. The VEGF/ VPF concentration in the cyst fluid obtained from patients who required repeated aspiration or underwent surgical resection because of recurrent accumulation (84.8 +/- 58.3 ng/mL, mean +/- SD, n = 18) was significantly higher than that in the cysts that regressed or disappeared after a single aspiration (4.3 +/- 4.4 ng/mL, n = 12, P < 0.001). These in vitro and clinical findings suggest that VEGF/VPF is at least partly involved in the accumulation of cyst fluid in thyroid nodules, and that a high VEGF/VPF concentration predicts rapid accumulation of the cyst fluid, possibly necessitating interventional treatment.

Autocrine role of insulin-like growth factor (IGF)-I in a human thyroid cancer cell line.

An established cell line (TC-cell, clone 78) derived from human thyroid papillary cancer cells was investigated for production of peptide growth factors. The cells had specific binding sites for insulin-like growth factor-I (IGF-I) and responded to this growth factor with increased proliferation. Culture medium conditioned by TC cells was found to contain insulin-like growth factor (IGF)-I and IGF-binding protein(s). Furthermore, reverse transcription-polymerase chain reaction revealed expression of IGF-I mRNA. When monoclonal antibody to IGF-I receptors (alpha IR3) was added, the growth of TC cells cultured in serum-free medium was significantly reduced. The growth rate of the cells was restored when the antibody was removed from the medium. These results strongly suggest that TC cells produce IGF-I, which is involved in the regulation of their own growth.

Monoclonal antibody immunohistochemistry of rabbit olfactory receptor neurons during development.

Staining patterns for monoclonal antibodies produced against the rabbit olfactory bulb were studied from embryonic day 14 up to 30 days after birth in the rabbit olfactory receptor neurons. One monoclonal antibody, 112D5, stained all of the receptor neurons in the olfactory epithelium and vomeronasal organ during development. The other, 114G12, showed a unique gradual expression in the olfactory receptor neurons. 114G12-Positive cells first appeared in the epithelium of the embryonic day-17 fetus. At embryonic day 25 or 26, 114G12-positive cells were situated in the superficial receptor cell layer. The arrangement in the positive and negative receptor neurons was 'superficial-positive' and 'deep-negative'. Thereafter, a gradual increase in the number of 'superficial-positive' cells was accompanied by a decrease in the 'deep-negative' cells. These changes continued until postnatal day 30. The negativity of staining for monoclonal antibody 114G12 in the deep compartment was retained in the adult rabbit. The supporting cells and basal cells were monoclonal antibody 114G12-negative throughout development. This unique developmental pattern suggests that monoclonal antibody 114G12 is a useful probe for studies on neurogenesis in adult animal. In contrast to the olfactory epithelium, the vomeronasal organ was monoclonal antibody 114G12-negative throughout development. Thus monoclonal antibody 114G12 makes a molecular distinction between the olfactory receptor neurons and vomeronasal system.

Monoclonal antibody immunohistochemistry of degenerative and renewal patterns in rabbit olfactory receptor neurons following unilateral olfactory bulbectomy.

Degeneration and regeneration of olfactory receptor neurons were studied in adult rabbits by immunohistochemical procedures following unilateral olfactory bulbectomy. Staining patterns of the olfactory receptors of the lesioned side were compared with those of the intact side in the nasal septum at various postoperative periods (12h-6 months) following lesion. Monoclonal antibodies, produced against the rabbit olfactory bulb, were used as histochemical markers. A slight decrease in the number of olfactory receptor neurons occurred at 24 h after lesion. One monoclonal antibody 112D5 stained all receptor neurons including degenerating neurons, but the other 114G12 showed a rapid decrease in immunostaining so that 114G12-positive cells disappeared within 7 days after lesion. 114G12-positive cells reappeared at 4 weeks following lesion. By 3 months, 114G12-positive cells were arranged in a plane at the apical region of the superficial compartment of the receptor cell layer, suggesting a recapitulation of development pattern of the receptor neurons. Thereafter, the number of 114G12-positive cells increased progressively and the staining pattern of the olfactory epithelium was like that of control animals by 6 months. Monoclonal antibody 114G12 is thus the first marker that is not specific to olfactory neurons and can be used to characterize certain embryonic traits during the degeneration and regeneration of the olfactory epithelium in the adult mammal.

Monoclonal antibody immunohistochemistry of adult rabbit olfactory structures.

Immunohistochemical staining patterns of two monoclonal antibodies produced against the rabbit olfactory bulb were studied in adult rabbit olfactory structures. One monoclonal antibody 112D5 (monoclonal antibody 2D5) stained all of the olfactory receptor cells, whereas the other 114G12 (monoclonal antibody 4G12) stained the upper two-thirds to three-fourths of the receptor cell layer. The negative region in the receptor cell layer was designated the deep compartment. Neither monoclonal antibody stained the supporting cells, basal cells, or Bowman's glands. Monoclonal antibody 2D5 stained the olfactory nerve layer and glomeruli in the olfactory bulb, whereas monoclonal antibody 4G12 stained the whole of the olfactory bulb, particularly the glomeruli and the mitral cells. The piriform cortex was unstained by monoclonal antibody 2D5 whereas the highest immunoreaction to monoclonal antibody 4G12 was in layer Ia. Immunoblot analysis revealed that the molecular weight values of monoclonal antibody 4G12 antigens in the olfactory epithelium were approx. 26,000. Thus, monoclonal antibody 4G12, specific to neurons, recognized an epitope different from the olfactory marker protein specific to the olfactory receptor neurons.

Subdivisions of the guinea-pig accessory olfactory bulb revealed by the combined method with immunohistochemistry, electrophysiological, and optical recordings.

The presence of subgroups in vomeronasal sensory neurons has been known in various animals. To elucidate possible functional subdivisions in the guinea-pig accessory olfactory bulb, the combined studies with GTP-binding protein immunohistochemistry, electrophysiological and optical recordings were carried out. Gi2 alpha and Go alpha proteins were immunohistochemically localized, respectively, in the anterior and posterior regions of the vomeronasal nerve and glomerular layers, indicating that the guinea-pig accessory olfactory bulb receives at least two different inputs. This suggests that an anatomical boundary exists in these two layers. A mapping study of field potentials in sagittal slice preparations demonstrated that stimulation of the anterior vomeronasal nerve layer elicited field potentials with weak oscillatory responses exclusively in the anterior region of the external plexiform layer, whereas shocks to the posterior vomeronasal nerve layer provoked distinct oscillatory responses within the posterior one. The damping factors of oscillations in the anterior and posterior regions were 0.064+/-0.028 and 0.025+/-0.014, respectively. These electrophysiological results suggest that the accessory olfactory bulb consists of two functionally different subdivisions. Real-time optical imaging showed that anterior vomeronasal nerve layer shocks produced neural activity which spread horizontally from anterior to posterior only within the anterior region of the external plexiform and mitral cell layers, whereas shocks to the posterior vomeronasal nerve layer evoked periodic neural activity which spread horizontally from posterior to anterior only within the posterior region. Furthermore, the most posterior extent of the optical response evoked in the anterior region immediately adjoined the most anterior extent of that evoked in the posterior region. The maximal distance of signal propagation in the granule cell layer corresponded to that in the overlying external plexiform and mitral cell layers, indicating that the granule cell layer also has a similar boundary. Thus, these optical imaging studies not only demonstrated a precise boundary in each layer of the accessory olfactory bulb, which was positioned right beneath the boundary defined by GTP-binding protein immunohistochemistry, but also confirmed the observations from electrophysiological mapping that evoked field potentials are independently distributed in each of two subdivisions. The presence of the functional subdivision in each layer leads us to conclude that the accessory olfactory bulb in the guinea-pig is distinctly segregated into the anterior and posterior subdivisions, and to suggest that there are at least two different input output pathways in the vomeronasal system.

Novel subdivisions of the rat accessory olfactory bulb revealed by the combined method with lectin histochemistry, electrophysiological and optical recordings.

Wistaria floribunda agglutinin and peanut agglutinin were found to bind histochemically to the anterior and posterior regions, respectively, of the vomeronasal nerve and glomerular layers in the rat accessory olfactory bulb. Furthermore, Ricinus communis agglutinin showed strong binding to the anterior region of the vomeronasal nerve and glomerular layers, whereas it bound weakly and/or moderately to the rostral two-thirds of the posterior glomerular layer but not at all to the caudal one-third. This suggests that the posterior region is further divided into two subregions. An electrophysiological mapping study in sagittal slice preparations demonstrated that stimulation given within the anterior vomeronasal nerve layer elicited field potentials within the anterior region of the external plexiform layer, whereas shocks to the rostral two-thirds and the caudal one-third of the posterior vomeronasal nerve layer provoked field responses within the rostral two-thirds and within the caudal one-third of the posterior external plexiform layer, respectively, indicating that the posterior external plexiform layer is also divided into two subregions. Real-time optical imaging showed similar results as above, except that neural activity also spread into mitral cell layers. Furthermore, the most anterior and posterior ends of the neural activity evoked in the rostral two-thirds of the posterior region immediately adjoined the posterior border of that evoked in the anterior region and the anterior border of that evoked in the caudal one-third of the posterior region, respectively. Moreover, the granule cell layer was also found to have similar boundaries. Thus, optical imaging studies demonstrated individual precise boundaries of these subdivisions, which were positioned right beneath those defined by Ricinus communis agglutinin histochemistry. The presence of functional segregation in each layer leads us to conclude that there are at least three different input-output pathways in the rat vomeronasal system.

Convergence of olfactory and gustatory connections onto the endopiriform nucleus in the rat.

Electrical and optical recordings were made from slice preparations including the piriform and gustatory cortices. Electrical stimulation of the gustatory cortex evoked a characteristic field potential in the endopiriform nucleus. A field potential was induced in the endopiriform nucleus by stimulation of the piriform cortex. Voltage-sensitive dye studies showed that stimulation of the piriform cortex induced signal propagation from the piriform cortex to endopiriform nucleus, whereas stimulation of the gustatory cortex did the same from the gustatory cortex to endopiriform nucleus via the agranular division of the insular cortex. After stimulation of the endopiriform nucleus, optical signals propagated not only to the piriform cortex but also to the gustatory cortex via the agranular division of the insular cortex. The olfactory and gustatory pathways appeared to be reciprocally connected. Unit recordings indicated that olfactory and gustatory activity converged onto a single neuron of the endopiriform nucleus. It is suggested that the cortical integration of olfactory and gustatory information could modulate mechanisms involved in food selection and emotional reactions relating to the chemical senses.

Phorbol ester pretreatment attenuates the growth hormone (GH) response to GH-releasing factor in cultured rat pituitary cells.

The effect of phorbol ester pretreatment on rat (r) GH release induced by GH-releasing factor (GRF) or 8-bromo-cyclic (c)AMP was investigated using rat pituitary cells cultured in monolayers. Pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 h significantly suppressed the rGH release induced by GRF, but not that by 8-bromo-cAMP 20 h later; this suppressive effect of TPA was concentration-dependent from 8 to 160 nmol/l, and complete suppression was observed after pretreatment with 80-160 nmol TPA/l. Production of cAMP by pituitary cells stimulated with GRF was similarly attenuated in TPA-pretreated cells. The rGH responsiveness to GRF of these cells was fully recovered on prolonged culture (40 h), suggesting that the inhibitory effect of TPA is reversible. In contrast, pretreatment with GRF (5 nmol/l) resulted in suppression of the rGH response to subsequent exposure to GRF (5 nmol/l) or 8-bromo-cAMP (10 mmol/l), but not to TPA. These observations suggest that pretreatment with TPA modifies the rGH response to GRF at steps before the formation of cAMP.

Insertion/deletion polymorphism in intron 16 of the ACE gene and left ventricular hypertrophy in patients with end-stage renal disease.

We studied the relationship between polymorphism in intron 16 of the angiotensin-converting enzyme (ACE) gene and left ventricular (LV) hypertrophy in uremic patients treated with hemodialysis therapy. The LV parameters were not different for age-, hematocrit-, and blood pressure-matched patients in DD, ID, and II genotype groups. The most important factor for LV hypertrophy was systolic blood pressure, which correlated with the posterior wall thickness (r=0.35; P=0.001) and LV mass index (LVMI; r=0.23; P=0.032). Among nonhypertensive patients, the frequency of interventricular septum (IVS) hypertrophy (>12 mm) and hypertrophy in LVMI (>145 g/m2) was significantly greater in patients with the DD genotype than in I allele-positive (+) patients. The odds rate for IVS hypertrophy was 5.04 (95% confidence interval, 1.15 to 24.8). These data suggest that the DD genotype of the ACE gene polymorphism is a contributory factor for the development of LV hypertrophy in patients with end-stage renal disease (ESRD).