Combination gemcitabine plus S-1 versus gemcitabine plus cisplatin for advanced/recurrent biliary tract cancer: the FUGA-BT (JCOG1113) randomized phase III clinical trial.

BACKGROUND: Gemcitabine plus cisplatin (GC) is the standard treatment of advanced biliary tract cancer (BTC); however, it causes nausea, vomiting, and anorexia, and requires hydration. Gemcitabine plus S-1 (GS) reportedly has equal to, or better, efficacy and an acceptable toxicity profile. We aimed to confirm the non-inferiority of GS to GC for patients with advanced/recurrent BTC in terms of overall survival (OS). PATIENTS AND METHODS: We undertook a phase III randomized trial in 33 institutions in Japan. Eligibility criteria included chemotherapy-naïve patients with recurrent or unresectable BTC, an Eastern Cooperative Oncology Group Performance Status of 0 - 1, and adequate organ function. The calculated sample size was 350 with a one-sided α of 5%, a power of 80%, and non-inferiority margin hazard ratio (HR) of 1.155. The primary end point was OS, while the secondary end points included progression-free survival (PFS), response rate (RR), adverse events (AEs), and clinically significant AEs defined as grade ≥2 fatigue, anorexia, nausea, vomiting, oral mucositis, or diarrhea. RESULTS: Between May 2013 and March 2016, 354 patients were enrolled. GS was found to be non-inferior to GC [median OS: 13.4 months with GC and 15.1 months with GS, HR, 0.945; 90% confidence interval (CI), 0.78-1.15; P = 0.046 for non-inferiority]. The median PFS was 5.8 months with GC and 6.8 months with GS (HR 0.86; 95% CI 0.70-1.07). The RR was 32.4% with GC and 29.8% with GS. Both treatments were generally well-tolerated. Clinically significant AEs were observed in 35.1% of patients in the GC arm and 29.9% in the GS arm. CONCLUSIONS: GS, which does not require hydration, should be considered a new, convenient standard of care option for patients with advanced/recurrent BTC. CLINICAL TRIAL NUMBER: This trial has been registered with the UMIN Clinical Trials Registry (http://www.umin.ac.jp/ctr/index.htm), number UMIN000010667.

Evaluation of the progression of non-azotemic proteinuric chronic kidney disease in dogs.

Proteinuria is a recognized risk factor for progression of canine chronic kidney disease (CKD). However, the prognosis of non-azotemic proteinuric CKD in dogs has been studied only to a limited extent. Moreover, the degree to which proteinuria should be decreased to delay CKD progression remains unknown. The purposes of this study were (1) to identify factors associated with disease progression and (2) to investigate the degree of proteinuria, albuminuria, and blood pressure during the course of treatment associated with the progression using time-averaged urine protein:creatinine ratio (UPC) and urine albumin:creatinine ratio (UAC) in canine non-azotemic proteinuric CKD. Twenty-one dogs with non-azotemic proteinuric CKD were included in the study. High UPC and UAC were associated with CKD progression (P < .05). Time-averaged high UPC and UAC were significantly related to progression (P < .05). The cutoff values of these time-averaged parameters for predicting the progression were 4.1 and 2.0, respectively. In dogs with non-azotemic proteinuric CKD, more severe proteinuria and albuminuria were associated with progression. The present study suggests that because UPC ≥ 4.1 and UAC ≥ 2.0 during treatment were associated with a faster progression of non-azotemic proteinuric CKD, therapeutic intervention is warranted.

Fibroblast growth factor-23 as an early marker of CKD-mineral bone disorder in dogs: preliminary investigation.

OBJECTIVES: To examine the relationship between fibroblast growth factor-23 levels, chronic kidney disease severity and mineral metabolic disorders associated to chronic kidney disease in dogs. MATERIALS AND METHODS: Fifteen control and 75 chronic kidney disease dogs were retrospectively included. Serum fibroblast growth factor-23 concentration and other phosphate metabolite parameters were compared between controls and each International Renal Interest Society stage. Multiple regression analysis was performed to determine the predictors of fibroblast growth factor-23. RESULTS: Serum fibroblast growth factor-23 concentrations were significantly higher in dogs with IRIS stages 2, 3 and 4 chronic kidney disease than those in dogs in control group and with stage 1 and increased along with the severity of chronic kidney disease. Compared with control dogs, serum intact parathyroid hormone significantly increased from stage 2 and serum phosphorus concentrations increased in dogs with stage 4. In dogs with stage 2, fibroblast growth factor-23 levels significantly increased in those with hyperphosphatemia compared with those with normophosphatemia. While eight of 26 (30.8%) dogs with stage 2 developed hyperparathyroidism (intact parathyroid hormone>8.5 ng/L), 19 (73.1%) dogs with stage 2 had elevated fibroblast growth factor-23 levels above the reference range (>528 pg/mL). Log creatinine, log intact parathyroid hormone and log product of total calcium and phosphorus were independent predictors of log fibroblast growth factor-23. CLINICAL SIGNIFICANCE: This preliminary study suggests that canine fibroblast growth factor-23 might be involved in mineral metabolic disorders associated to chronic kidney disease in dogs, and this factor could be potentially used as an early marker for this condition.

Use of vertebral left atrial size for staging of dogs with myxomatous valve disease.

INTRODUCTION/OBJECTIVES: The American College of Veterinary Internal Medicine (ACVIM) guidelines suggest that pimobendan should be initiated in dogs which meet all criteria of stage B2 myxomatous mitral valve disease (MMVD): murmur intensity ≥ 3/6, left atrial-to-aortic ratio ≥ 1.6, normalized left ventricular internal diameter in diastole ≥ 1.7, and vertebral heart size > 10.5. Recently, a new radiographic index for left atrial enlargement, vertebral left atrial size (VLAS), was proposed. The objective of the present study was to evaluate whether VLAS is useful in staging MMVD and if it can distinguish between ACVIM stages B1 and B2. ANIMALS: Ninety-seven client-owned dogs with MMVD were evaluated and classified as ACVIM stage B1, B2, or C-D. MATERIALS AND METHODS: The echocardiographs and radiographs of all the dogs were retrospectively evaluated to obtain left atrial-to-aortic ratio, normalized left ventricular internal diameter in diastole, and VLAS values. The data were analyzed to assess the correlation between these measurements and VLAS, and the optimal cutoff value of VLAS was determined. RESULTS: A VLAS cutoff value of 2.6 provided the greatest diagnostic accuracy for identification of dogs with ACVIM stage B2 MMVD (area under the curve, 0.96; sensitivity, 95%; specificity, 84%). A VLAS ≥2.5 exhibited the highest sensitivity (sensitivity, 100%; specificity, 78%), and a VLAS ≥ 3.1 exhibited the highest specificity (sensitivity, 47%; specificity, 100%). CONCLUSIONS: VLAS is a helpful index for monitoring MMVD using radiography. A VLAS cutoff value of 2.5 could be used to identify dogs that may benefit from echocardiography to determine if they have reached ACVIM stage B2.

Identification of oral species of the genus Veillonella by polymerase chain reaction.

INTRODUCTION: Members of the genus Veillonella cannot be reliably distinguished by their biochemical characteristics and phenotypic features. Moreover, DNA-DNA hybridization and sequence analyses of the 16S ribosomal RNA gene including random fragment length polymorphism analysis, are complex and time-consuming procedures that are not well-suited to identifying oral species of Veillonella: Veillonella atypica, Veillonella denticariosi, Veillonella dispar, Veillonella parvula, and Veillonella rogosae. METHODS: In this study, five forward primers and a reverse primer were designed for polymerase chain reaction (PCR) according to the partial sequences of the rpoB genes of these oral Veillonella species. RESULTS: The forward primers were species-specific for these five Veillonella species, and could produce specific amplicons when used together with reverse primer and individual DNA templates of these species in PCR. These primer pairs were also found to discriminate between the respective species, and the Veillonella strains isolated from human oral cavities were successfully assigned to one of the five oral species of the genus Veillonella based on their specific products by PCR. CONCLUSION: A simple two-step PCR procedure using the five sets of primer pairs developed in the present study is a rapid and reliable method for the identification of the recognized oral Veillonella species.

Evaluation of the therapeutic results of epiduroscopic adhesiolysis for failed back surgery syndrome.

BACKGROUND: No data for patients with failed back surgery syndrome (FBSS) based on the location of adhesions separated by epiduroscopic adhesiolysis have been reported. METHODS: We performed epiduroscopic adhesiolysis on 28 FBSS patients to examine the impact of differences in the locations of the separated regions on the treatment results. We performed fluoroscopic imaging through the sacral hiatus to assess the condition of adhesions in the epidural space during the post-adhesiolysis observation period. RESULTS: In patients in whom only the epidural space was separated by adhesiolysis, there was a significant improvement in the Roland-Morris disability questionnaire (RDQ) score until 12 weeks after adhesiolysis, but the score gradually returned to the preoperative value thereafter. Among patients in whom the nerve root responsible for radicular pain was separated, there was a long-term improvement in the RDQ, Oswestry disability index 2.0 (ODI), and Japanese Orthopedic Association Assessment of Treatment (JOA) scores. Among patients in whom both the epidural space and the nerve root responsible for pain were separated, there was a 12 week improvement in the RDQ score and 24 week improvements in the ODI and JOA scores. CONCLUSIONS: Progressive epidural imaging after adhesiolysis suggested that pain was caused by re-adhesion around the nerve root. Since re-adhesion of the nerve root required some time, the effect of adhesiolysis was maintained for extended periods in these cases. We suggest that epiduroscopic adhesiolysis is an effective therapy for FBSS patients, and that adhesiolysis of the nerve root may exhibit the long-term (24 weeks) efficacy in patients with pain.

Synaptically activated increases in Ca2+ concentration in hippocampal CA1 pyramidal cells are primarily due to voltage-gated Ca2+ channels.

Changes in intracellular Ca2+ concentration ([Ca2+]i) in the soma and dendrites of hippocampal CA1 pyramidal neurons were measured using intracellularly injected fura-2. A large component of the [Ca2+]i elevation caused by high frequency stimulation of the Schaffer collaterals was correlated with the Na+ spikes triggered by the excitatory postsynaptic potentials (EPSPs). These spikes were generated in the soma and proximal dendrites and stimulated Ca2+ entry through voltage-gated Ca2+ channels. Suppressing spikes by hyperpolarizing the soma or by injecting QX-314 revealed a smaller nonspike component of Ca2+ entry. A substantial fraction of this component was mediated by the action of the EPSPs on voltage-gated Ca2+ channels, because it persisted in 2-amino-5-phosphonovaleric acid and because it was usually reduced when Ca2+ channel activity was suppressed by hyperpolarization. Ca2+ entry through the N-methyl-D-aspartate receptor channel could not be detected with certainty, perhaps because it was highly localized.

Primary biliary cirrhosis and autoantibodies.

Fifty years have passed since anti-mitochondrial antibodies were found in patients with primary biliary cirrhosis (PBC). PBC is an autoimmune hepatic disease in which 85-90% of patient antibodies bind to mitochondrial antigens that include pyruvate dehydrogenase complex (PDC)-E2 and other members of the oxaloacid dehydrogenase family. In addition, indirect immunofluorescence (IIF) assays utilizing HEp-2 cell substrates have been used to identify anti-centromere antibodies in 20-30% of PBC sera. These antibodies are generally easily recognized, however, anti-nuclear envelope and anti-multiple nuclear dot antibodies are occasionally more difficult to recognize with certainty by IIF. The use of enzyme linked immunosorbent assays that utilize recombinant gp210 (an autoantigen of the nuclear envelope) and/or sp100 (a protein target represented by multiple nuclear dots) should be particularly considered in anti-mitochondrial antibody negative PBC sera. Although the clinical significance of these antibodies still remains to be determined, there is evidence that the existence of anti-gp210 antibodies are related to poorer prognosis and more aggressive disease progression.

Updated results from GEST study: a randomized, three-arm phase III study for advanced pancreatic cancer.

PURPOSE: The GEST study showed non-inferiority of S-1 but not superiority of gemcitabine plus S-1 (GS) to gemcitabine alone for overall survival with the data by the cut-off date of 31st July in 2010 for chemo-naïve patients with advanced pancreatic cancer. We considered it important to determine whether S-1 maintains non-inferiority after a long-term follow-up in the GEST study and to obtain a firm positive conclusion. In addition, it may be an interesting challenge to explore the efficacious profile of GS in the long-term follow-up study. Using the data from the follow-up period, background and efficacy in patients from Taiwan and Japan, as well as the rates of tumor shrinkage in locally advanced and metastatic patients (Waterfall plot) were also analyzed. METHODS: The results of the primary analysis were reconfirmed, and subset analysis of overall survival and progression-free survival was performed based on the overall survival data updated by the cut-off date of 31st July in 2011. RESULTS: The median follow-up period was 29.8 months, and 795 deaths occurred (95.6%). The median overall survival was 8.8 months for gemcitabine, 9.7 months for S-1 (hazard ratio [HR], 0.96; 97.5% confidence interval [CI], 0.79-1.17), and 9.9 months for GS (HR 0.91; 97.5% CI 0.75-1.11). In patients with performance status (PS) 0, the median overall survival was 9.8 months for gemcitabine, 10.9 months for S-1, and 10.5 months for GS. In patients with PS 1, the median overall survival was 6.2 months for gemcitabine, 6.3 months for S-1, and 9.6 months for GS. CONCLUSION: Our survey reconfirmed the non-inferiority of S-1 to gemcitabine and showed S-1 can be used as one of the standard treatment options for advanced pancreatic cancer. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00498225.

Identification of the second tonicity-responsive enhancer for the betaine transporter (BGT1) gene.

When certain cells are exposed to a hypertonic solution, transcription of the BGT1 gene is markedly increased. The ensuing rise in betaine transport leads to cellular accumulation of betaine that protects the cells from the stress of hypertonicity. We have previously identified a tonicity-responsive enhancer (TonE1) in the 5' flanking region of the BGT1 gene. It was recognized, however, that full activation of transcription requires additional sequence upstream from the TonE1. Now we report that there is another TonE (named TonE2) 72 base pairs upstream from the TonE1. TonE1 and TonE2 act synergistically to stimulate transcription of BGT1 in response to hypertonicity.

Direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the CA1 region of the rat hippocampus.

Recent studies have demonstrated that astrocytes express a variety of ion channels and neurotransmitter receptors and can modulate the activity of neurons. Since a single astrocyte makes tight contacts with many neighboring neuronal cells, they can provide efficient and wide modulation of neuronal networks. Here, we provide direct evidence for mutual interactions between perineuronal astrocytes and interneurons in the stratum radiatum of the rat hippocampus. Direct depolarization of a perineuronal astrocyte suppressed the excitatory postsynaptic currents in an adjacent interneuron and increased the paired-pulse ratio, indicating that perineuronal astrocytes have a suppressive effect on presynaptic elements. Moreover, perineuronal astrocyte activation modulated the directly induced firing pattern of the interneuron, with initial facilitation and subsequent suppression. Conversely, direct firing of the interneuron depolarized the membrane potential and reduced the input resistance of the perineuronal astrocyte. These results directly demonstrate the existence of bidirectional interactions between neurons and perineuronal astrocytes.

Optical monitoring of synaptic summation along the dendrites of CA1 pyramidal neurons.

The primary function of neurons is to integrate synaptic inputs and to transmit the results to other cells. Recent studies with somatic whole-cell recordings have shown that separate excitatory inputs to hippocampal or cortical pyramidal neurons are summated non-linearly. In the present study, we examined how postsynaptic potentials (PSPs) are summated along the dendrites employing fast optical voltage imaging techniques. Rat hippocampal slices were stained with a fluorescent voltage-sensitive dye (JPW1114) and optical signals were monitored with a 16 x 16 photodiode array system. Two independent input pathways were stimulated individually or in pairs through glass electrodes such that different locations of the dendrites received separate synaptic inputs. We found that (1) the summation of PSPs was sub-linear along the entirety of dendrites, (2) the blockade of GABA(A) receptors suppressed sub-linearity and (3) further blockade of GABA(B) receptors suppressed sub-linearity of the summation of separate inputs on apical dendrites. Our study demonstrates that pyramidal neurons integrate PSPs linearly along the entirety of dendrites; moreover, GABAergic inputs are responsible for maintaining sub-linear summation in CA1 pyramidal neurons.

Optical detection of dendritic spike initiation in hippocampal CA1 pyramidal neurons.

Previous studies have shown that spikes can be generated in the dendrites of CA1 pyramidal neurons. Some have suggested that, in response to synaptic inputs, spikes are initiated near the soma and propagate back into the dendrites, but some recent studies have shown that intense synaptic inputs initiate spikes in the dendrite. Here, we report the optical detection of spike propagation along the apical dendrites of hippocampal pyramidal neurons. Rat hippocampal slices were stained with the fluorescent voltage-sensitive dye, JPW1114, and optical signals monitored using a 16 x 16 photodiode array system at a frame rate of 4 kHz. A stimulating electrode was placed at the boundary between the stratum (str.) lacnosum-moleculare and the str. radiatum to stimulate the Schaffer collateral, and fast and slow signal components were detected in the dendritic and somatic regions. By comparing the optical signals with whole-cell recordings, we confirmed that the fast component was due to a population of dendritic spikes in pyramidal neurons. The fast component appeared in dendritic locations near the input sites in response to synaptic activation, and signal onset at the soma was delayed by a few milliseconds compared with that at the input sites. Local perfusion of a Na(+) channel blocker near the soma eliminated the fast component at the soma, but had no effect on the fast component at the input sites. Our results indicate that dendritic spikes can be initiated in dendrites near the input site and propagate orthodromically toward the proximal dendrites and the soma.

Adverse effects of an active fragment of parathyroid hormone on rat hippocampal organotypic cultures.

Adverse effects of an active fragment of parathyroid hormone (PTH(1 - 34)), a blood Ca(2+) level-regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 microM PTH(1 - 34) for 60 min resulted in a gradual increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH(1 - 34) at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose-dependent over the range of 0.1 pM to 0.1 microM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH(39 - 84), or by an active fragment of PTH-related peptide (PTHrP(1 - 34)), an intrinsic ligand of the brain PTH receptor. The PTH(1 - 34)-induced adverse effects were significantly inhibited by co-administration of 10 microM nifedipine, an L-type Ca(2+) channel blocker, but not by co-administration of blockers of the other types of Ca(2+) channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca(2+) overloading via activation of dihydropyridine-sensitive Ca(2+) channels, and suggests that PTH may be a risk factor for senile dementia. British Journal of Pharmacology (2000) 129, 21 - 28

Detection of anti-branched chain 2-oxo acid dehydrogenase complex (BCOADC)-E2 antibody in primary biliary cirrhosis by ELISA using recombinant fusion protein.

Anti-M2 of anti-mitochondrial antibody (AMA) is a serological marker of primary biliary cirrhosis (PBC). Anti-pyruvate dehydrogenase complex-E2 (anti-PDC-E2) is recognized as the most frequently occurring anti-M2, and a routine laboratory test for this antibody has already been established. However, it is also known that there are patients with PBC who are negative for anti-PDC-E2. For the serological diagnosis of these patients, immunoblotting for anti-M2s is indicated. However, the technique currently utilized is too laborious to allow testing of a large number of samples. In this study, we have developed an enzyme-linked immunosorbent assay (ELISA) using a recombinant fusion protein in order to evaluate anti-branched chain 2-oxo-acid dehydrogenase complex-E2 (anti-BCOADC-E2), another frequently occurring anti-M2 in PBC patients. KB cell lines (CCL 17) were utilized as source material, and BCOADC-E2 cDNA (971 bp) including the lipoic acid binding domain was amplified by polymerase chain reaction. The amplified region was subcloned into pEX-3 vectors and expressed, and the resulting fusion protein (beta-galactosidase/BCOADC-E2) was utilized as antigen for an ELISA. We ascertained the specificity of this antigen by inhibition tests with ELISA and immunoblotting. We defined the cut-off optical density (OD) value as the mean + 3 SD (0.146) of sera from 60 normal controls. Anti-BCOADC-E2 could not be detected with this assay in sera from normal controls and from patients with autoimmune hepatitis and chronic viral hepatitis. Anti-BCOADC-E2 was detected in 119 of 210 sera (56.7%) from patients with PBC. In addition, anti-BCOADC-E2 was detected in 48 of 99 (48.5%) sera from PBC patients who were negative for anti-PDC-E2. Here, we have succeeded in developing a new ELISA for detecting anti-BCOADC-E2. This system is antigen-specific and easily performed. This assay should allow routine testing of a large number of serum samples, and should become especially useful for the serodiagnosis of anti-PDC-E2-negative PBC patients.

Immunoreactivity to various human cytochrome P450 proteins of sera from patients with autoimmune hepatitis, chronic hepatitis B, and chronic hepatitis C.

Numerous human Cytochrome P450 enzymes (CYPs) associated with 'phase I' drug metabolism have been identified. Among them, CYP2D6 is thought to be the major target autoantigen to anti-liver kidney microsome (LKM)-1 autoantibody, a characteristic feature of autoimmune hepatitis (AIH) type II. In this study, we were able to clone CYP2D6 cDNA from a human liver cDNA library and express the CYP2D6 recombinant protein, and also to prepare four other representative human CYP proteins (CYP1A2, 2C9, 2E1, and 3A4). These preparations were used to assay the immunoreactivity of patients with AIH type I (n=35) and type II (n=9). As comparison groups, sera from patients with chronic hepatitis B (n=15), chronic hepatitis C (n=55; 24 anti-LKM-1-positive, 31 anti-LKM-1-negative), and from normal controls (n=30) were included. The five CYP proteins did not react with sera from normal controls nor from patients with chronic hepatitis B. CYP2D6 reacted with sera from 100% (9/9) of AIH type II patients, 79% (19/24) of patients with anti-LKM-1-positive chronic hepatitis C, and 6.5% (2/31) of patients with anti-LKM-1-negative chronic hepatitis C. In contrast, CYP1A2 reacted with serum from one patient with AIH type I, CYP2E1 reacted with sera from two patients with AIH type I, one patient with anti-LKM-1-positive chronic hepatitis C, and two patients with anti-LKM-1-negative chronic hepatitis C, and CYP3A4 reacted with sera from one patient with AIH type II and one patient with anti-LKM-1-positive chronic hepatitis C. CYP2C9 did not react with any of the sera included in this study. From these results, it is suggested that CYPs other than CYP2D6 can function as immunotargets in certain disease conditions.

Detection of anti-LKM-1(anti-CYP2D6) by an enzyme-linked immunosorbent assay in adult patients with chronic liver diseases.

Anti-liver kidney microsome-1 (LKM-1) autoantibody, which is a serological marker for autoimmune hepatitis type II, recognizes Cytochrome P450 IID6 (CYP2D6). This autoantibody is also detected in a portion of patients with chronic hepatitis C. Anti-LKM-1 has been measured by indirect immunofluorescence (IF) using rat liver and kidney sections. However, this method has some problems in specificity and is so laborious to handle with many samples. In this study, in order to determine anti-LKM-1, we established an enzyme-linked immunosorbent assay (ELISA) for anti-CYP2D6 using a recombinant CYP2D6 fusion protein. We studied sera from 29 patients positive for anti-LKM-1 by the new ELISA. We further studied sera from a total of 301 patients with various liver diseases and 100 sera from normal controls negative for anti-LKM-1 by the new ELISA. The specificity of the ELISA was ascertained by absorption tests using sera positive for anti-LKM-1. In 29 sera from patients positive for anti-LKM-1 by IF, we found a good correlation between the logarithms of the antibody titers determined by IF and ELISA indexes obtained by our new method. Anti-CYP2D6 was positive in 12 of 12 (100%) patient with autoimmune hepatitis type II and 16 of 17(94.1%) with chronic hepatitis C positive for anti-LKM-1 by IF. In other 401 sera negative for anti-LKM-1 by IF, anti-CYP2D6 was all negative except a few sera. We established a new ELISA for anti-LKM-1 (anti-CYP2D6). This ELISA system is sensitive, antigen-specific and easy to be done. Therefore, this assay allows a routine test of many serum samples, especially for diagnosing autoimmune hepatitis type II.

Effects of glucocorticoids and cyclosporine on IL-2 and I kappa B alpha mRNA expression in human peripheral blood mononuclear cells.

To evaluate molecular mechanisms that might account for the heterogeneity in the in vitro responsiveness of individual subjects' peripheral blood mononuclear cells (PBMC) to immunosuppressive drugs, the authors quantitated in normal human cells the suppressive effects of the glucocorticoids prednisolone and methylprednisolone and of cyclosporine on interleukin-2 (IL-2) mRNA expression and IL-2 production, as well as the stimulatory effect of these drugs on IkappaBalpha mRNA expression. As expected, cyclosporine was significantly more suppressive than either glucocorticoid of IL-2 mRNA expression and IL-2 production by mitogen-stimulated PBMC, with variable degrees of inhibition in cells from individual subjects. The authors confirmed in human PBMC the stimulation of IkappaBalphamRNA expression by the glucocorticoid reported by others in HeLa and transfected Jurkat cell lines. In addition, the authors observed a stimulatory effect on IkappaBalpha mRNA expression by cyclosporine as well in 8 of 10 PBMC preparations studied, suggesting a possible role of calcineurin in the regulation of IkappaBalpha production. Interindividual variability in the intracellular mechanisms of action, possibly based on molecular polymorphisms, might be one factor contributing to differences among patients in their clinical responses to treatment with such drugs.

A hyperpolarizing response induced by glutamate in mouse cerebellar Purkinje cells.

In the vertebrate nervous system, glutamate (Glu) receptors are generally known to cause depolarizing responses. We report here a novel type of Glu response in Purkinje neurons of mouse cerebellar slices, namely glutamate-induced hyperpolarization (GH). This response is not due to activation of inhibitory interneurons, because application of tetrodotoxin (TTX), bicuculline, or strychnine did not abolish GH. In addition, GH persisted in a Ca(2+)-free or a low-Cl- solution, which rules out the involvement of gK(Ca) or GABAA mechanisms. Quisqualate (Quis) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), which are potent and selective agonists, respectively, for the metabotropic Glu receptor (mGluR), failed to induce GH. L-2-Amino-4-phosphonobutyric acid (L-AP4) was also ineffective. Simultaneous recording of electrical activity and intracellular Ca2+ concentration ([Ca2+]i) showed that GH was not accompanied by [Ca2+]i changes. Voltage clamp experiments showed that GH is due to reduction of a tonically active conductance with a reversal potential around 0 mV. Two possible mechanisms are suggested for GH: (1) changes in the desensitized steady state of ionotropic Glu receptors, or (2) a novel Glu-mediated mechanism.

High time resolution fluorescence imaging with a CCD camera.

We have built a high speed, sensitive camera system capable of capturing sequences of low-light level images synchronized with recordings of membrane potential. The camera system is based on a cooled, scientific grade CCD camera controlled by a PC/AT computer. It can take 100 frames/sec of 18 X 18 element images and 40 frames/sec of 50 X 50 element images with no lag in response to step changes in light intensity. High accuracy and dynamic range of the measurements result from the fact that light levels of the picture elements are digitized with 12 bit accuracy with intrinsic camera noise levels typically less than 1/10,000 of the maximum detectable light level. We have used this system to record calcium dependent fura-2 fluorescence transients in the dendrites of cerebellar Purkinje cells and from different regions of leech neurons in segmental ganglia or isolated in culture.