Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1.

Triaxial testing system for pressure core analysis using image processing technique.

In this study, a newly developed innovative triaxial testing system to investigate strength, deformation behavior, and/or permeability of gas hydrate bearing-sediments in deep sea is described. Transport of the pressure core from the storage chamber to the interior of the sealing sleeve of a triaxial cell without depressurization was achieved. An image processing technique was used to capture the motion and local deformation of a specimen in a transparent acrylic triaxial pressure cell and digital photographs were obtained at each strain level during the compression test. The material strength was successfully measured and the failure mode was evaluated under high confining and pore water pressures.

Differential changes in messenger RNA expressions and binding sites of neuropeptide Y Y1, Y2 and Y5 receptors in the hippocampus of an epileptic mutant rat: Noda epileptic rat.

The anti-convulsive effects of neuropeptide Y have been suggested in several animal models of epilepsy. We have found the sustained increase of neuropeptide Y contents and the seizure-induced elevation of hippocampal messenger RNA in a novel spontaneous epileptic mutant rat: Noda epileptic rat. In the present study, we investigated the change of neuropeptide Y Y1 and Y2 receptor messenger RNA expressions and binding sites in the hippocampus following a spontaneous generalized tonic-clonic seizure of Noda epileptic rat. Furthermore, the binding sites of a more recently isolated receptor subtype, neuropeptide Y Y5 receptors, were also evaluated by receptor autoradiography. A marked elevation of neuropeptide Y immunoreactivity in the mossy fiber, and Y2-receptor up-regulation in the dentate gyrus were observed in the hippocampus of Noda epileptic rat, which coincided with the previous results of the other epileptic models. In contrast, Y1-receptor down-regulation was not found after a spontaneous seizure of Noda epileptic rat while this occurs in kindling and after kainic acid-induced seizures. [125I][Leu31, Pro34]peptide YY/BIBP 3226-insensitive (Y5 receptor) binding sites in CA1 stratum radiatum were significantly decreased following a spontaneous seizure of Noda epileptic rat. The present results suggest that a spontaneous seizure of Noda epileptic rat induces significant changes in neuropeptide Y-mediated transmission in the hippocampus via Y2 and Y5 receptors, but not Y1 receptors. Therefore, specific subset of neuropeptide Y receptor subtypes might be involved in the epileptogenesis of Noda epileptic rat.

Successful in vitro generation of Epstein-Barr virus-specific cytotoxic T lymphocytes from severe chronic active EBV patients.

Severe chronic active Epstein-Barr virus (EBV) infection (SCAEBV) is a rare but life-threatening disorder. Poor cytotoxic activity against the virus is widely believed to contribute to the development of this disease. We wished to determine whether it is possible to generate autologous EBV-specific cytotoxic T cells (CTLs) in vitro that can be infused back into the patient to treat his/her viremia. To do this, we first had to establish autologous EBV-transformed B cells (EBCL) as antigen-presenting cells, which is known to be difficult to do with B cells from SCAEBV patients. In one patient, the standard method of incubating B cells with EBV-containing B95-8 supernatant was sufficient. In a second patient, however, the B cells apoptosed too rapidly in culture to permit transformation. However, apoptosis could be blocked by the presence of CD40 ligand-transfectant cells, and EBV transformation was successful when performed with this transfectant. Indicating a native immune response to EBV, peripheral blood lymphocytes from both patients proliferated in response to autologous EBCL. Furthermore, patient T cells had higher frequencies of IFN-gamma-producing CD8 cells after stimulation with autologous EBCL than sero-positive healthy controls. EBV-specific CTLs could be generated from both patients after repeated stimulation with autologous EBCL. These CTL lines were predominantly composed of CD4+ cells, and autologous EBCL killing was largely inhibited by an antibody against HLA-DR. These findings support the possibility of adoptive immune therapy to treat SCAEBV patients.

Paroxysmal kinesigenic choreoathetosis locus maps to chromosome 16p11.2-q12.1.

Paroxysmal kinesigenic choreoathetosis (PKC), the most frequently described type of paroxysmal dyskinesia, is characterized by recurrent, brief attacks of involuntary movements induced by sudden voluntary movements. Some patients with PKC have a history of infantile afebrile convulsions with a favorable outcome. To localize the PKC locus, we performed genomewide linkage analysis on eight Japanese families with autosomal dominant PKC. Two-point linkage analysis provided a maximum LOD score of 10.27 (recombination fraction [theta] =.00; penetrance [p] =.7) at marker D16S3081, and a maximum multipoint LOD score for a subset of markers was calculated to be 11.51 (p = 0.8) at D16S3080. Haplotype analysis defined the disease locus within a region of approximately 12.4 cM between D16S3093 and D16S416. P1-derived artificial chromosome clones containing loci D16S3093 and D16S416 were mapped, by use of FISH, to 16p11.2 and 16q12.1, respectively. Thus, in the eight families studied, the chromosomal localization of the PKC critical region (PKCR) is 16p11.2-q12.1. The PKCR overlaps with a region responsible for "infantile convulsions and paroxysmal choreoathetosis" (MIM 602066), a recently recognized clinical entity with benign infantile convulsions and nonkinesigenic paroxysmal dyskinesias.

Familial paroxysmal kinesigenic choreoathetosis: an electrophysiologic and genotypic analysis.

PURPOSE: We report a pedigree of familial paroxysmal kinesigenic choreoathetosis (PKC) in which five of 18 members are affected. The pathophysiologic basis for PKC is still uncertain; reflex epilepsy versus dysfunction of basal ganglia. We examined (a) whether there were ictal discharges during the attacks, and (b) a linkage between PKC and possible DNA markers linked to several familial epileptic or movement disorders. METHODS: Video-monitoring EEG was performed in two patients with PKC during attacks elicited by movements of the lower extremities. Blood samples for DNA studies were obtained from 15 members of the pedigree. Fourteen polymorphic markers on chromosomes 1p, 2q, 6p, 10q, and 20q were genotyped, and two-point lod scores were calculated for each marker under a dominant model. RESULTS: No ictal discharges were found during the attacks in both patients. We could not obtain significant linkage of PKC with any marker examined. CONCLUSIONS: The video-monitoring EEG findings in our cases strongly suggested that the etiology of PKC should be considered distinct from that of reflex epilepsy. However, the patients in this pedigree had experienced generalized convulsions in their infancies; thus we could not deny the possibility of an epileptogenic basis for PKC. There was no strong evidence for a linkage of the gene for PKC with the candidate regions on 1p, 2q, 6p, 10q, or 20q.

Elevated neuropeptide Y and corticotropin-releasing factor in the brain of a novel epileptic mutant rat: Noda epileptic rat.

Noda epileptic rat (NER) is a new epileptic rat strain, which was developed by inbreeding rats with spontaneous tonic-clonic seizures in a stock of Crj:Wistar. In the present study, possible changes of two neuropeptides, neuropeptide Y (NPY) and corticotropin-releasing factor (CRF), in the brains of NER were investigated. Increased contents of immunoreactive (IR) NPY were found in the striatum and amygdala of 8-week NERs with partial seizure, while these changes extended to the limbic region including hippocampus in 16-week NERs with fully developed generalized tonic-clonic seizure. IR-CRF were elevated only in the entorhinal and pyriform cortex of both 8-week and 16-week NERs. Generalized tonic-clonic seizure in NERs induced a transient increase of NPY mRNA in the granular layer of dentate gyrus. These results suggest that NPY metabolism in the limbic brain contributes to the seizure susceptibility in this model of epilepsy.

Trimethyltin syndrome as a hippocampal degeneration model: temporal changes and neurochemical features of seizure susceptibility and learning impairment.

The effects of trimethyltin on the hippocampus were investigated in terms of changes in histology, depth electroencephalography, learning acquisition and memory retention, choline acetyltransferase and neuropeptides, and seizure-induced c-fos messenger RNA expression. The results were as follows. (1) Morphologically, trimethyltin produced a progressive loss of hippocampal CA3 and CA4 pyramidal cells, starting from four days after peroral treatment with trimethyltin hydroxide (9 mg/kg), as described previously. (2) Neurophysiologically, the increased seizure susceptibility to pentylenetetrazol treatment reached a maximum at four days post-trimethyltin and then declined after five days post-trimethyltin. The maximal seizure susceptibility at four days post-trimethyltin was confirmed by the immediate and long-lasting appearance of spike discharge in the hippocampus. However, this was not verified by the expression of c-fos messenger RNA in the hippocampus, which was comparable between trimethyltin-treated and control rats. (3) Behaviorally, the time-courses of aggression and learning impairment were similar to that of the seizure susceptibility. (4) Neurochemically, trimethyltin treatment caused changes of neurochemical markers, which were manifested by the elevation of neuropeptide Y content in the entorhinal cortex, and of choline acetyltransferase in the hippocampal CA3 subfield. Trimethyltin may offer potential as a tool for investigations on the relationship between neuronal death in the hippocampus and the development of seizure susceptibility and learning impairment. Alterations in glucocorticoids, glutamate and neuropeptides may all contribute to the manifestation of the trimethyltin syndrome.

Resolution of late potentials with improvement in left ventricular systolic function in patients with first acute myocardial infarction.

BACKGROUND: Ventricular late potentials predict subsequent arrhythmic events and sudden death in postinfarction patients. Late potentials are recorded in the infarcted area, but it should be pointed out that they probably represent micropotentials in the area of delayed conduction found among isolated areas of scar tissue and normal myocardium. HYPOTHESIS: The study was undertaken to investigate the relationship between chronic reversible myocardial ischemia, such as stunned or hibernating myocardium, and late potentials in 38 patients with a first myocardial infarction. METHODS: The patients were divided into two groups, one with (Group 1) and one without (Group 2) resolution of late potentials recorded by signal-averaged electrocardiogram at 6 months after onset of myocardial infarction. We investigated the clinical, echocardiographic, and signal-averaged electrocardiographic characteristics of Groups 1 and 2. RESULTS: In the chronic phase of myocardial infarction, a higher incidence of patency of the infarct-related artery and an improvement of wall motion score were found in Group 1, and left ventricular ejection fraction was better preserved in Group 1 than in Group 2. CONCLUSIONS: These results suggest that the resolution of late potentials was influenced by the improvement of left ventricular systolic function and patency of the infarct-related artery. We concluded that chronic reversible myocardial ischemia, such as stunned or hibernating myocardium, might be the substrate that generated late potentials.

Changes of immunoreactive neuropeptide Y, somatostatin and corticotropin-releasing factor (CRF) in the brain of a novel epileptic mutant rat, Ihara's genetically epileptic rat (IGER).

Ihara's genetically epileptic rat (IGER) is a rat mutant with genetically scheduled spontaneous convulsions mimicking human limbic seizures. In the present study, the possible changes of three neuropeptides, neuropeptide Y (NPY), somatostatin (SRIF) and corticotropin-releasing factor (CRF), in the brains of IGER were investigated. Increased contents of immunoreactive (IR) NPY were found only in the hippocampus of 2-month IGERs before developing convulsive seizures, while similar increases of IR-NPY were discovered in the striatum and pyriform and entorhinal cortex as well as hippocampus in 8-month IGERs with repetitive seizures. There were no significant differences in the brain contents of IR-SRIF and IR-CRF between IGERs and the controls at both ages. These findings indicate an enhanced rate of NPY synthesis in this experimental model of epilepsy which may play a critical role in the development of epileptogenesis.

Sustained ventricular tachycardias associated with myotonic dystrophy.

Patients with myotonic dystrophy are reported to have a higher frequency of sudden death than the general population. Although causes of sudden death in myotonic dystrophy are suggested to be due to conduction of defects progressing, the HV interval cannot predict whether conduction system disease would develop or progress. We report two cases of myotonic dystrophy complicated with sustained monomorphic ventricular tachycardias (VT), which can cause sudden death. In Case No. 1, although the patient was treated successfully for sustained VT with verapamil in electrophysiologic studies, another sustained VT was confirmed 2 years later. In Case No. 2, the patient showed decreased left ventricular ejection fraction and late potentials, and induced sustained VT that was identical to clinically documented VT. Although VT is believed to be rare in patients with myotonic dystrophy, these cases suggest that VT is a possible cause of sudden death.

Prevalence, resolution, and determinants of late potentials in patients with unstable angina and left ventricular wall motion abnormalities.

Although transient myocardial ischemia such as exercise-induced ischemia has not been reported to be associated with the occurrence of late potentials, the association of late potentials with more profound ischemic damage, which is represented by reversible but prolonged left ventricular wall motion abnormalities, has not been demonstrated. We prospectively evaluated 37 unstable angina patients who had reversible but prolonged wall motion abnormalities after resolution of chest pain and electrocardiogram (ECG) changes. Signal-averaged ECG (SAECG) and echocardiogram were recorded during the acute phase and before hospital discharge. Late potentials were present in 6 (16 percent) patients on the initial SAECG recording and resolved in all 6 patients on the second recording before hospital discharge. Normalization of inferior left ventricular wall motion abnormality and multivessel disease were observed more frequently in patients with late potentials on the initial recording than in patients without (p < 0.05 and p < 0.05, respectively). In conclusion, late potentials were observed in patients who had reversible but prolonged wall motion abnormalities; these late potentials were resolved with improvement of left ventricular wall motion abnormalities. These results suggest that myocardial ischemia with prolonged wall motion abnormalities is a possible mechanism of the occurrence of late potentials.

Nitric oxide synthase-containing neurons in the hippocampus are preserved in trimethyltin intoxication.

We studied the effects of trimethyltin (TMT) (9 mg/kg, p.o.) on the nitric oxide synthase (NOS)-containing neurons in the rat hippocampus by NADPH-diaphorase histochemistry and a biochemical assay of NOS activity. TMT exposure caused the typical behavioral changes and a loss of the CA3/4 pyramidal cells, which were NADPH diaphorase-negative. The scattered interneurons and the CA1 pyramidal cells, which were NADPH diaphorase-positive, were spared. Hippocampal NOS activity showed no reduction in the TMT-treated rats compared with the controls. These results provide evidence of the preservation of the NOS-containing neurons in TMT intoxication.

Altered brain contents of neuropeptides in spontaneously epileptic rats (SER) and tremor rats with absence seizures.

Immunoreactive- (IR-) somatostatin (SRIF), neuropeptide Y (NPY) and corticotropin-releasing factor (CRF) contents were investigated in the brain of tremor rats with absence-like seizure and spontaneously epileptic rats (SER), which is a genetically defined double-mutant (zi/zi, tm/tm) obtained by mating zitter homozygote (zi/zi) with tremor heterozygote (tm/+) and shows both absence-like seizure and tonic convulsions. Increased levels of IR-NPY and IR-CRF were observed in several regions including the amygdala and hippocampus in homozygous SER compared to heterozygous SER (zi/zi, tm/+ or +/+). Homozygous tremor rats (tm/tm) showed lower levels of IR-NPY and IR-CRF contents mainly in the hippocampus and mesolimbic system (entorhinal and pyriform cortex and nucleus accumbens) than heterozygous tremor rats. IR-SRIF contents of homozygous SER were higher in frontal cortex than heterozygous SER and in amygdala than homozygous tremor rats. No change of IR-SRIF between groups was noted in the hippocampus among brain structures underlying epileptogenicity. The results suggest that the change of neuropeptide levels, most conspicuous in NPY among three peptides tested, may be involved in the phenotypical manifestation of seizures in SER and tremor rats, and that the development of tonic convulsion and absence seizures may be differently associated with the change of brain neuropeptide levels.

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.

The effect of successful angioplasty on variables of signal-averaged electrocardiogram and ventricular wall motion in patients with a first myocardial infarction.

The correlation among three variables of late potentials (LPs) obtained by signal-averaged electrocardiography (SAECG) and improvement of ventricular wall motion estimated by echocardiography were studied in 66 patients with a first acute myocardial infarction (MI). Patients with bundle-branch block, intraventricular conduction delay, multi-vessel disease, previous MI, repeat percutaneous transluminal coronary angioplasty (PTCA), or evidence of reinfarction during a 6-month follow-up were excluded. A total of 66 patients was divided into two groups, with (Group 1: n = 27, age 56 +/- 11) or without (Group 2: n = 39, age 61 +/- 10) improvement of ventricular wall motion. Three variables of LPs and ventricular wall motion index (WMI) estimated and scored by echocardiography at admission (WMI 1) and at 6 months after MI (WMI 2) were compared in each group. In Group 1 (WMI 1 vs. WMI 2, p < 0.002), 20 of 27 patients underwent successful angioplasty; in Group 2 (WMI 1 vs. WMI 2, p = NS), 7 of 39 patients had successful emergency angioplasty. There were significant differences in three variables of LPs between the time of admission and at 6 months after MI in Group 1 but not in Group 2. Higher incidence of LPs and greater frequency of successful emergency PTCA were found in Group 1 compared with Group 2. These results suggest that because myocardial ischemia is reversed by successful angioplasty, ventricular wall motion is improved and the arrhythmogenic substrate that generates LPs is stabilized electrically. Stunned or hibernating myocardium may be the arrhythmogenic substrate that generates LPs.

Effect of body characteristics on the variables of signal-averaged electrocardiograms in healthy subjects.

Late potentials have been reported to be affected by body size or left ventricular mass. To our knowledge, however, the effect of subadipose tissue, which is known to influence QRS amplitudes of the surface ECG on the variables of late potentials, has not been evaluated. The relationships between the variables of late potentials and various obesity indices were assessed in 45 men, aged 24 to 38 years, without structural heart disease and bundle branch blocks. QRS duration (DUR), root mean square voltage in the last 40 ms (RMS), and low-amplitude signals < 40 microV (LAS) were obtained by signal-averaged ECG. Left ventricular mass (LV mass) was determined by echocardiography. The DUR and RMS had no correlation with body height, weight, body mass index (BMI), sum of skin folds (triceps and subscapular), or LV mass. Positive linear correlations were found between LAS and weight (r = 0.48, p < 0.002), BMI (r = 0.54, p < 0.002), sum of skin folds (r = 0.57, p < 0.002), and percent BMI (r = 0.54, p < 0.002). Subadipose tissue may shift the onset of the 40-microV point of LAS to the left with a consequent prolongation of LAS by attenuation of the QRS complex. These data suggest that the use of LAS alone or as a combination in an obese population for the definition of positive late potentials is inappropriate.

Late potentials during left ventricular healing of acute myocardial infarction.

BACKGROUND: Late potentials and left ventricular remodeling are important factors in the prognosis of acute myocardial infarction. However, the relationship between late potentials and ventricular remodelling has not been fully evaluated. METHODS: We evaluated clinical characteristics, coronary angiographic findings and radionuclide angiographic measures about 1 month after an acute myocardial infarction in patients with and without late potentials. RESULTS: Although the left ventricular ejection fraction of patients with late potentials was not different from that of patients without late potentials, the left ventricular end-diastolic volume of patients with late potentials was larger than that of patients without late potentials (P < 0.05). There was a significant positive correlation between the left ventricular end-diastolic volume and the filtered QRS duration (r = 0.53, P < 0.001). The root mean square of the voltage in the terminal 40 ms and the low-amplitude signal duration of < 40 microV in the terminal QRS sequence were also correlated with the left ventricular end-diastolic volume (r = 0.40, P < 0.02, and r = 0.39, P < 0.02, respectively). Patency of the infarct-related vessel in the late phase of an acute myocardial infarction was an important factor associated with the occurrence of late potentials (P < 0.01). CONCLUSION: A larger left ventricular end-diastolic volume in patients with late potentials might be associated with left ventricular remodeling during the first month after an acute myocardial infarction.

A novel synthetic phyllolitorin analogue [desTrp3,Leu8]phyllolitorin inhibits scratching behavior induced by neuromedin C in rats.

[DesTrp3,Leu8]phyllolitorin (DTP) (pGlu-Leu-Ala-Val-Gly-Ser-Leu-Met-NH2) was synthesized as an analogue of phyllolitorins, a new member of bombesin family, and examined if it antagonized neuromedin C (NMC)-induced scratching. DTP inhibited dose-dependently the scratching behavior by NMC (1 microgram), whereas it did not alter any element of other grooming behaviors. DTP (6 micrograms) alone was found to be neither toxic nor active in inducing both scratching and grooming, which were comparable to vehicle alone. Assuming that the scratching behavior is commonly and specifically elicited by bombesin family peptides, DTP might be classified as a new type of bombesin antagonist.

The effect of delta sleep-inducing peptide (DSIP) on the changes of body (core) temperature induced by serotonergic agonists in rats.

Hyperthermia induced by high doses of 5-methoxy-N,N-dimethyl-tryptamine (5-MeODMT) was diminished and hypothermia induced by low doses of 5-MeODMT was enhanced by pretreatment with delta sleep-inducing peptide (DSIP). Delta sleep-inducing peptide had an enhancing effect of hypothermia induced by 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). This action of DSIP was completely inhibited by ICV injection of anti-DSIP. Pindolol prevented the enhancing action of DSIP on both 8-OH-DPAT- and apomorphine-induced hypothermia. It is suggested that the thermoregulatory action of DSIP is primarily exerted by a 5-HT1A mechanism in the rat.