Intracellular recording of identified neostriatal patch and matrix spiny cells in a slice preparation preserving cortical inputs

1. The morphology, electrical membrane properties, and corticostriatal excitatory postsynaptic potentials (EPSPs) of two groups of neostriatal projection cells, patch cells, and matrix spiny cells were compared in the rat by the use of an in vitro slice preparation that preserves inputs from medial agranular cortex. Spiny cells were stained intracellularly with biocytin and identified as belonging to the patch (striosomal) compartment or to the matrix by immunohistochemistry for the 28 kD calcium-binding protein calbindin on the same sections. 2. Patch and matrix neurons had very similar axonal and dendritic morphology. Both patch and matrix cells extended their dendrites and local axon collaterals almost exclusively in their respective compartments. Patch cells and most matrix cells had local axon collaterals within or near the parent dendritic domain. However there was a class of matrix cells that extended axon collaterals over a much wider portion of the neostriatum but still restricted to the matrix compartment. 3. Input resistance and membrane time constant were estimated from the membrane response to intracellularly applied current pulses. The average values of matrix cells were and 8.41 ms. The values of patch cells were 31.8 M omega and 8.19 ms and were within the range of those of matrix cells. Both types of cells showed the same kinds of membrane nonlinearities when tested with the use of current pulses. Input resistance and time constant were both strongly affected by a fast anomalous rectification and were thus voltage-dependent, decreasing with membrane polarization. Slow ramplike depolarizing responses were observed in response to depolarizing current steps. 4. Repetitive firing was examined with the use of depolarizing current pulses. In both types of spiny cells, trains of action potentials showed little adaptation of spike frequency and linearly increased with current intensities less than 1 nA. The slopes frequency, calculated from the first and second intervals, were 115.0 and 107.2 Hz/nA, respectively, for matrix cells and 86.0 and 82.8 Hz/nA for patch cells. 5. Stimulation of the medial agranular cortex induced EPSPs in some striatal cells in both compartments. EPSP in matrix cells often showed both short-latency and long-latency components, corresponding to two early components of the response observed in vivo. Some matrix cells, and all patch cells, showed only the longer latency EPSP component. The average latency was 6.3 ms in matrix cells and 9.1 ms in patch cells. The relationship between EPSP amplitude and membrane potential was nonlinear, with EPSP amplitude and duration increasing with decreasing membrane polarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Acute effect of inhaled beta(2)-agonists with different type of intrinsic activity on airway resistance in healthy volunteers]

Inhaled beta(2)-agonists (long-acting as well as short acting) are used world-wide for the relief of asthma symptoms. However, there are few reports which have evaluated the additive effect of short-acting beta(2)-agonists to long-acting beta(2)-agonists on airway resistance measured by a plethysmography. This study was designed to evaluate the additive effect of inhaled short-acting beta(2)-agonists (protecarol) to long-acting beta(2)-agonists (salmeterol) on airway resistance in normal healthy volunteers (S+P group). In addition, to compare the effects of beta(2)-agonists which have different types of intrinsic activities, acute effect of inhaled procaterol adding to procaterol was also evaluated (P+P group). Seven healthy volunteers (all male and all non-smokers) were entered in this study. Pulmonary function was measured by a body plethysmography. Forced expiratory volume per 1 second (FEV1), the maximum flow rate at 25% (V(.) 25), the maximum flow rate at 50% of forced vital capacity (V(.) 50), and airway resistance were measured before and after inhalation of salmeterol (1 dry powder, 50 microg) or procaterol (2 puffs, 20 microg). Sixty minutes after inhalation of salmeterol, or 15 minutes after inhalation of procaterol, inhalation of procaterol (2 puffs, 20 microg) was added, and then pulmonary function was monitored. FEV1, V(.) 25, and V(.) 50 were significantly increased after inhalation of salmeterol as well as procaterol. In addition, airway resistance decreased significantly after inhalation of salmeterol as well as procaterol. In the S+P group, additional decrease of airway resistance after inhalation of procaterol was relatively small compared with the P+P group. In conclusion, although additional bronchodilatoric effects were observed in the S+P and P+P group, the effects seemed to be different based on the intrinsic activity of each beta(2)-agonist.     

ENCEPHALOMYELONEURITIS WITH MEDIASTINAL GERM CELL TUMOR A Paraneoplastic Condition ?

An unusual case of encephalomyeloneuritis associated with germ cell tumor with mature and immature teratoma arising in the mediastinum is presented. There was an unusually long interval from the onset of neurologic symptoms to the development of malignancy. The histopathology, characterized by limbic encephalitis, brain stem encephalitis, cortical cerebellar degeneration and myeloneuritis, was similar to that of paraneoplastic encephalomyeloneuritis previously described in the literature. Virological and immunological studies failed to demonstrate any causative agents or autoantibodies reacting with brain tissue. The causal relationship between the malignant neoplasm and encephalomyeloneuritis thus seems to be very complex.     

Large aspiny cells in the matrix of the rat neostriatum in vitro: physiological identification, relation to the compartments and excitatory postsynaptic currents.

1. Large aspiny neurons (20-60 microns diam) in the neostriatum were studied in an in vitro rat slice preparation by whole-cell recording to reveal physiological identification from medium-sized spiny projection cells (10-20 microns diam), relation to the patch and matrix compartments, and excitatory synaptic inputs. Recorded cells were identified by intracellular biocytin staining. Compartmental identification was made by calbindinD28K immunohistochemistry in fixed slices. 2. Large stained neurons were morphologically heterogeneous and had aspiny or sparsely spiny dendrites and dense local axonal branches. They were located in the matrix or on the patch-matrix border. Axonal branches of the large aspiny cells were preferentially distributed in the matrix and gave off terminal boutons there. Some of the secondary dendrites arising from stem dendrites running along the border, however, crossed compartment boundaries and made fine branches in a patch. 3. Large aspiny cells had less negative resting membrane potentials and lower thresholds for spike generation than medium spiny cells. They showed longer-duration and larger-amplitude afterhyperpolarizations (AHPs) than medium spiny cells. During hyperpolarizing current pulses, apparent resistance slowly reduced, and a prominent sag was observed in the voltage record, which was absent in medium spiny cells. The large aspiny cells showed no spontaneous firing but had a tendency to fire repetitive spikes in response to depolarizing current pulses, although spike interval tended to increase in later spikes. Spike frequency of large aspiny cells increased less with current intensity than that of medium spiny cells. 4. Most large aspiny cells were considered to belong to a single physiological class, although one large aspiny cells showed shorter-duration AHPs than both most other large aspiny cells and medium spiny cells, and little spike-frequency adaptation. 5. Excitatory postsynaptic currents (EPSCs) of large aspiny cells induced by intrastriatal stimulation had two components. An early, linear component was blocked by 10 microM 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a selective antagonist of non-N-methyl-D-aspartate (NMDA) receptors. A later component with a nonlinear current-voltage (I-V) relationship was blocked by 50 microM DL-2-amino-5-phosphonovaleric acid (DL-APV), a selective antagonist of NMDA receptors. 6. From these results, four conclusions can be drawn. 1) Most large aspiny neostriatal cells in the matrix, although they take heterogeneous shapes, belong to one physiological class with long-duration AHPs and a strong time-dependent component of anomalous rectification.(ABSTRACT TRUNCATED AT 400 WORDS)     

The plasminogen activation system reduces fibrosis in the lung by a hepatocyte growth factor-dependent mechanism

Mice deficient in the plasminogen activator inhibitor-1 gene (PAI-1-/- mice) are relatively protected from developing pulmonary fibrosis from bleomycin administration. We hypothesized that one of the protective mechanisms may be the ability of the plasminogen system to enhance hepatocyte growth factor (HGF) effects, which have been reported to be anti-fibrotic in the lung. HGF is known to be sequestered in tissues by binding to extracellular matrix components. Following bleomycin administration, we found that HGF protein levels were higher in bronchoalveolar lavage fluid from PAI-1-/- mice compared to wild-type (PAI-1+/+) mice. This increase could be suppressed by administering tranexamic acid, which inhibits plasmin activity. Conversely, intratracheal instillation of urokinase into bleomycin-injured PAI-1+/+ mice to activate plasminogen caused a significant increase in HGF within bronchoalveolar lavage and caused less collagen accumulation in the lungs. Administration of an anti-HGF neutralizing antibody markedly increased collagen accumulation in the lungs of bleomycin-injured PAI-1-/- mice. These results support the hypothesis that increasing the availability of HGF, possibly by enhancing its release from extracellular matrix by a plasmin-dependent mechanism, is an important means by which activation of the plasminogen system can limit pulmonary fibrosis.     

Cell lines that support replication of a novel herpes simplex virus 1 UL31 deletion mutant can properly target UL34 protein to the nuclear rim in the absence of UL31

Previous results indicated that the herpes simplex virus 1 (HSV-1) U(L)31 gene is necessary and sufficient for localization of the U(L)34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U(L)31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Vero cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U(L)31 gene. The replication of the U(L)31 deletion virus was restored on U(L)31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U(L)34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U(L)34 protein localized at the nuclear membrane in rabbit skin cells, and U(L)31 complementing CV1 cells infected with the U(L)31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U(L)34 protein to the nuclear membrane. We speculate that this function partially complements that of U(L)31 and may explain why U(L)31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.     

Antitumor immune response by CX3CL1 fractalkine gene transfer depends on both NK and T cells

The CX3C chemokine fractalkine (CX3CL1) exists as both a membrane-bound form promoting firm cell-cell adhesion and a soluble form chemoattracting leukocytes expressing its receptor CX3CR1. When adenoviral vector expressing mouse fractalkine (AdFKN) was transduced to the tumor cells, fractalkine was expressed as both membrane-bound form on the tumor cells and soluble form in the supernatant in vitro. Intratumoral injection of AdFKN (1 x 10(9)PFU/tumor) into C26 and B16F10 tumors resulted in marked reduction of tumor growth compared to control (C26: 86.5%, p<0.001; B16F10: 85.5%, p<0.001). Histological examination of tumor tissues revealed abundant infiltration of NK cells, dendritic cells, and CD8(+) T lymphocytes 3 and/or 6 days after treatment with AdFKN. Splenocytes from mice treated by AdFKN developed tumor-specific cytotoxic T cells, and thereby protected from rechallenging with parental tumor cells. Antitumor effects by AdFKN were completely abrogated in both NK cell-depleted mice and CD8(-/-) mice, and partially blocked in CD4(-/-) mice. These data indicated that fractalkine mediates antitumor effects by both NK cell-dependent and T cell-dependent mechanisms. This study suggests that fractalkine can be a suitable candidate for immunogene therapy of cancer because fractalkine induces both innate and adaptive immunity.     

Diagnostic utility of galectin-3 and CD26/DPPIV as preoperative diagnostic markers for thyroid nodules

The aim of this study was to search for diagnostic markers that could correctly identify thyroid nodular lesions requiring urgent surgical treatment. We investigated whether galectin-3 and dipeptidyl peptidase IV (CD26/DPPIV) could be potential markers for improving the diagnostic accuracy of conventional cytology. Seventy-nine patients with histologically proven thyroid diseases were analyzed. The immunocytochemical staining results showed galectin-3 expression in neoplastic cells of all 37 papillary carcinomas, five of six follicular carcinomas, all three anaplastic carcinomas, one of three medullary carcinomas, and two of 14 follicular adenomas. All 16 adenomatous goiters were negative for galectin-3 immunostaining. On the other hand, all 37 papillary carcinomas, all six follicular carcinomas, and one of three anaplastic carcinomas revealed CD26/DPPIV expression, whereas all three medullary carcinomas were negative. Among benign thyroid lesions, four of 14 follicular adenomas and two of 16 adenomatous goiters exhibited varying degrees of immunoreactivity for CD26/DPPIV. RT-PCR analysis demonstrated overexpression of galectin-3 and CD26/DPPIV mRNAs in all six papillary and all three follicular carcinomas analyzed, whereas the mRNA expressions of these molecules were barely or not detectable in benign thyroid lesions and normal thyroid tissues, except for one case of follicular adenoma. In conclusion, we demonstrate that galectin-3 and CD26/DPPIV were consistently coexpressed at protein and mRNA levels in differentiated thyroid carcinomas. We propose that combined immunostaining for galectin-3 and CD26/DPPIV in the preoperative evaluation of thyroid nodules may play a role in accurate cytodiagnosis.     

Neuron-specific enolase and Leu-7 immunoreactive small round-cell neoplasm. The relationship to Ewing's sarcoma in bone and soft tissue

The authors present an immunohistochemical analysis of tissue from five cases of morphologically distinctive Ewing's sarcoma in bone and soft tissue. The mean age of the five patients was 16.6 years, with a range of 6-28 years. The tumors existed in chest wall, pelvis, and lower extremities. Two siblings with tumor in bone were clinically diagnosed as having Ewing's sarcoma. All cases had immunoreactivity for neuron-specific enolase (NSE), and four cases revealed positive staining for Leu-7. Neuron-specific enolase is highly specific for neurons and neuroendocrine cells. In addition, immunoreactivity for Leu-7, expressing a natural killer activity, has been demonstrated in peripheral nerve fibers and neuroendocrine cells. The authors suggest that NSE and Leu-7 immunoreactive small round-cell neoplasm is probably a primitive neuroectodermal tumor and should be categorized as Ewing's sarcoma in bone and soft tissue.