Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer.

To develop gene-modified T-cell-based antileukemia adoptive immunotherapy, concomitant administration of CD4(+) and CD8(+) T cells that have been gene modified using identical HLA class I-restricted leukemia antigen-specific T-cell receptor (TCR) gene transfer has not yet been fully investigated. Here, using CD4(+) and CD8(+) T cells that had been gene modified with a retroviral vector expressing HLA-A*24:02-restricted and Wilms' tumor 1 (WT1)-specific TCR-α/ß genes and siRNAs for endogenous TCRs (WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells), we examined the utility of this strategy. WT1-siTCR/CD4(+) T cells sufficiently recognized leukemia cells in an HLA class I-restricted manner and provided target-specific Th1 help for WT1-siTCR/CD8(+) T cells. By using a xenografted mouse model, we found that WT1-siTCR/CD4(+) T cells migrated to leukemia sites and subsequently attracted WT1-siTCR/CD8(+) T cells via chemotaxis. Therapy-oriented experiments revealed effective enhancement of leukemia suppression mediated by concomitant administration of WT1-siTCR/CD4(+) T cells and WT1-siTCR/CD8(+) T cells. Importantly, this augmented efficacy in the presence of WT1-siTCR/CD4(+) T cells was correlated with longer survival and enhanced formation of memory T cells by WT1-siTCR/CD8(+) T cells. Collectively, our experimental findings strongly suggest that this strategy would be clinically advantageous for the treatment of human leukemia.

Tunable terahertz emission from the intrinsic Josephson junctions in acute isosceles triangular Bi2Sr2CaCu2O8+δ mesas.

In order to determine if the mesa geometry might affect the properties of the coherent terahertz (THz) radiation emitted from the intrinsic Josephson junctions in mesas constructed from single crystals of the high-temperature superconductor, Bi2Sr2CaCu2O8+δ, we studied triangular mesas. For equilateral triangular mesas, the observed emission was found to be limited to the single mesa TM(1,0) mode. However, tunable radiation over the range from 0.495 to 0.934 THz was found to arise from an acute isosceles triangular mesa. This 47% tunability is the widest yet observed from the outer current-voltage characteristic branch of such mesas of any geometry. Although the radiation at a few of the frequencies in the tunable range appear to have been enhanced by cavity resonances, most frequencies are far from such resonance frequencies, and can only be attributed to the ac-Josephson effect.

Effects of N-acyl homoserine lactone analogues on Porphyromonas gingivalis biofilm formation.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobic rod Porphyromonas gingivalis in oral biofilms is a primary etiological agent of periodontal disease. Biofilm formation of various gram-negative bacteria is regulated by a quorum-sensing circuit that relies on N-acyl homoserine lactones (HSLs). Some synthetic N-acyl HSL analogues act as quorum-sensing inhibitors and suppress biofilm formation in Pseudomonas aeruginosa. Development of chemical control agents against oral biofilms is necessary, because until now, biofilms have been removed only by mechanical debridement. The present study investigated the effect of N-acyl HSL analogues on P. gingivalis biofilm formation, with the aim of developing new drugs that inhibit oral biofilm formation. MATERIAL AND METHODS: A flow-cell model was used for P. gingivalis biofilm formation. Seventeen synthetic N-acyl HSL analogues were quantitatively assessed by spectrophotometry. The effects of three antagonistic compounds against P. gingivalis biofilm formation were further examined by confocal laser scanning microscopy, and investigated for primary attachment using spectrophotometry and phase contrast microscopy. RESULTS: Ten out of 17 analogues affected P. gingivalis biofilm formation. Three out of 10 analogues significantly decreased biofilm-forming cells (p < 0.05), and these biofilm structures were less well formed three-dimensionally. There were no quantitative or qualitative differences in cell attachment between the control and the three analogue-treated groups. CONCLUSION: Three synthetic N-acyl HSL analogues inhibited biofilm formation in P. gingivalis. We suggest that these analogues influence the development stage of P. gingivalis biofilm formation.

Statin-independent prognosis of patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP therapy.

BACKGROUND: A recent laboratory study indicated that statins impaired the antitumor effects of rituximab by inducing conformational changes in CD20. Although these findings raised significant concerns about statin use during rituximab treatment, their clinical significance is unclear. PATIENTS AND METHODS: We conducted a retrospective study investigating the effects of statins on the prognosis of diffuse large B-cell lymphoma (DLBCL) treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP). Newly diagnosed DLBCL patients were analyzed (n = 256), including 35 patients taking statins. RESULTS: The 3-year progression-free survival rates were 84% and 73% (P = 0.38), while the overall survival rates were 89% and 78% (P = 0.28) for those patients treated with and without statins, respectively. After adjusting for the International Prognostic Index and serum cholesterol level, statin use was not associated with prognosis. CONCLUSIONS: These results indicate that statins do not influence the clinical prognosis of DLBCL treated with RCHOP. Further studies with larger numbers of patients are warranted to confirm the prognostic significance of statins for patients with DLBCL receiving rituximab-containing chemotherapy.

Functional difference of receptor-type protein tyrosine phosphatase zeta/beta isoforms in neurogenesis of hippocampal neurons.

Receptor-type protein tyrosine phosphatase zeta/beta (RPTPzeta) is a transmembrane chondroitin sulfate proteoglycan (CSPG) and has been shown to play crucial roles in controlling axonal growth and neuronal migration. The RPTPzeta has two transmembranous isoforms, shorter receptor form of RPTPzeta (sRPTPzeta) and full-length receptor form of RPTPzeta (fRPTPzeta), but no studies have been reported about functional difference of these two isoforms. In the present study, therefore, we examined whether or not two RPTPzeta isoforms have different role in controlling dendritic morphology and synaptic number in cultured hippocampal neurons using the quantitative morphometrical analysis. Confocal microscopic observation showed that the immunoreactivity of RPTPzeta was observed throughout cells such as axons, growth cones, and dendrites at the early stages of neuronal culture, while it was seen predominantly on dendrites at the late stages. Western blotting analysis revealed that fRPTPzeta was mainly expressed at the early stages of culture and both RPTPzeta isoforms were expressed at late stages of culture. The overexpression of sRPTPzeta in hippocampal neurons increased the dendritic arborization without altering the average length of dendritic branches, whereas that of fRPTPzeta decreased the dendritic arborization and increased the average length of dendritic branches. The RNA interference of fRPTPzeta expression increased the dendritic arborization without altering the average length of dendritic branches. The overexpression of fRPTPzeta decreased the density of hippocampal dendritic synapses, but that of sRPTPzeta had no effects. Pleiotrophin, a ligand for RPTPzeta to interfere the phosphatase activity, increased the density of hippocampal dendritic synapses. Thus, the present study demonstrates that two transmembranous RPTPzeta isoforms have different functions for regulating dendritogenesis and synaptogenesis.

Soluble interleukin-2 receptor retains prognostic value in patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP (RCHOP) therapy.

BACKGROUND: Soluble interleukin-2 receptor (SIL-2R) is known to be a prognostic parameter in patients with diffuse large B-cell lymphoma (DLBCL) receiving cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP) therapy. However, its prognostic value has not been well known since the introduction of rituximab. PATIENTS AND METHODS: We retrospectively evaluated the prognostic impact of SIL-2R in 228 DLBCL patients, comparing 141 rituximab-combined CHOP (RCHOP)-treated patients with 87 CHOP-treated patients as a historical control. RESULTS: Patients with high serum SIL-2R showed significantly poorer event-free survival (EFS) and overall survival (OS) than patients with low SIL-2R in both the RCHOP group (2-year EFS, 66% versus 92%, P<0.001; OS, 82% versus 95%, P=0.005) and the CHOP group (2-year EFS, 40% versus 82%; OS, 61% versus 90%, both P<0.001). Multivariate analysis including the five parameters of International Prognostic Index (IPI) and two-categorized IPI revealed that SIL-2R was an independent prognostic factor for EFS and OS in the RCHOP group as well as in the CHOP group. CONCLUSIONS: Our results demonstrate that SIL-2R retains its prognostic value in the rituximab era. The prognostic value of SIL-2R in DLBCL patients receiving rituximab-combined chemotherapy should be reassessed on a larger scale and by long-term follow-up.

CD5 expression is potentially predictive of poor outcome among biomarkers in patients with diffuse large B-cell lymphoma receiving rituximab plus CHOP therapy.

BACKGROUND: Several biomarkers indicating poor prognosis have been reassessed in patients receiving rituximab combination chemotherapy for diffuse large B-cell lymphoma (DLBCL). However, few studies have investigated outcome in relation to a combination of these biomarkers. In addition, no large-scale studies have reassessed the outcome of patients with CD5-positive DLBCL treated with rituximab. PATIENTS AND METHODS: We conducted a retrospective study and investigated the predictive value of three biomarkers -- BCL2, germinal center (GC) phenotype and CD5 -- in 121 DLBCL patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone. RESULTS: CD5-positive patients showed significantly poorer event-free survival (EFS) and overall survival (OS) than CD5-negative patients (2-year EFS, 18% versus 73%, P < 0.001; 2-year OS, 45% versus 91%, P = 0.001). However, no significant difference in outcome according to BCL2 or GC phenotype was observed. Multivariate analysis revealed that CD5 expression was a significant prognostic factor for EFS [hazard ratio 14.2, 95% confidence interval (CI) 4.7-43.2] and OS (hazard ratio 20.3, 95% CI 3.6-114.4). CONCLUSIONS: CD5 expression was the only significant prognostic factor among the biomarkers examined in this study. Further studies with larger numbers are warranted to confirm the prognostic significance of CD5 expression for patients with DLBCL receiving rituximab-containing chemotherapy.

Effect of zaprinast on nitric oxide levels in serum and aortic tissue.

Nitric oxide (NO), which is synthesized from the guanidino nitrogen of l-arginine by nitric oxide synthase (NOS), plays an important role in many physiological and pathological processes. Most of the effects of NO are mediated by cyclic guanosine 3'5 monophosphate (cGMP), which is synthesized by soluble guanylate cyclase (sGC) and degraded by phosphodiesterases (PDEs). Although the NO/cGMP pathway has been extensively studied, remarkably little is known about the regulation of NO release. Furthermore, controversial studies have indicated that intervention of the sGC/cGMP pathway modulates the release of NO. The purpose of this study was to evaluate the hypothesis that drugs that affect the sGC/cGMP pathway may modulate NO release and, if so, is there a correlation between NO levels and blood pressure effect? To this end, we investigated the effects of the PDE 5 inhibitor zaprinast on mean arterial pressure (MAP), nitrite/nitrate levels and cGMP in normotensive male Sprague Dawley rats. The results of the current study indicated that zaprinast dose-dependently increased plasma cGMP levels at 18, 24 and 36 mg/kg and decreased MAP at 24 and 36 mg/kg. However, zaprinast at 18, 24 and 36 mg/kg did not affect NO levels either in serum or aortic tissue. We have concluded that the PDE 5 inhibitor zaprinast has no regulatory effect on NO release in serum and aortic tissue, and NO was not involved in the hypotensive effect of zaprinast.

Establishment of specific pathogen-free (SPF) rat colonies using gnotobiotic techniques.

Gnotobiotic Wistar rats were produced using gnotobiotic techniques, which were established in the production of a SPF mouse colony, in order to establish a barrier-sustained colony. One strain of Escherichia coli, 28 strains of Bacteriodaceae (B-strains), three strains of Lactobacillus (L-strains) and a chloroform-treated fecal suspension (CHF, Clostridium mixture) were prepared from conventional Wistar rats as the microflora source. Two groups of limited-flora rats, E. coli plus B-strains and E. coli plus CHF, were produced. After confirmation that Clostridium difficile was not detected in the CHF-inoculated rats, two groups of limited-flora rats were transferred to an isolator and housed together in a cage. These rats were then orally inoculated with L-strains. The gnotobiotic rats showed colonization resistance to Pseudomonas aeruginosa, and the number of E. coli in the feces was 10(5) to 10(6)/g. The gnotobiotic rats were transferred to a barrier room as a source of intestinal flora for SPF colonies. In the SPF rats, basic cecal flora was mainly composed of Bacteroidaceae, clostridia, fusiform-shaped bacteria and lactobacilli, and did not change over a long period. Their flora became similar to that of conventional rats.

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.

Modality of postural movement in men and women with both arms flexed during standing.

We investigated postural movement associated with bilateral arm flexion in response to a light signal during standing in 179 healthy men and women to assess whether individual and sex differences arc evident in the postural movement pattern. The following results were obtained. (a) A correlation of -.87 was noted between movement angles of the foot-leg and leg-trunk. (b) Individual differences in movement angle were approximately twice as large in the hip joint as in the ankle and knee joints, and the movement angle of the leg trunk showed approximately half the number of extension as flexion movements. (c) The postural movement pattern was categorized on the basis of the movement angle of the foot leg and leg trunk into the following three patterns: hip flexion, backward leaning, and hip extension. The percentages of subjects showing these patterns were 59.2%, 33.5%, and 7.3%, respectively. (d) The inclination angle reflecting the righting response showed a gradual increase in size in the order of trunk, head, and neck. However, the righting response was not controlled precisely enough to enable subjects to maintain the inclination angle in a quiet standing posture. (e) We identified a significant sex difference in the relative frequency of subjects in the postural movement pattern.

Morphologically extracted trabecular skeleton superimposed upon digital radiograph structure.

The purpose of this study was to employ a morphological filter to digital X-ray images to extract the morphology of trabecular structures in a clearly understandable visual format. This study compares the trabecular skeleton extracted by a morphological filter to the original digital radiographic image by superimposing the images. A morphological filter (a combination of a single structuring element and a skeleton operation) based on a mathematical morphology theory was used to extract the skeletal pattern of trabecular bone from a digital X-ray image of a human femoral neck. Subset images with different operation numbers (n = 1, n = 2) were obtained, and then each image was superimposed on the original digital radiographic image using the superimpose function of a workstation. The extracted trabecular skeleton pattern was fairly consistent with the trabecular structure seen on the digital image according to the opinion of seven dentomaxillofacial radiologists. In their opinion, the majority of the structural elements were reproduced on the extracted skeleton. However, accurate skeleton elements were not extracted in the region of dense trabecular structure. The morphological filter was able to extract a large portion of the bone trabecular structure as a binary skeletal pattern image from trabecular bone on digital X-ray image, but more work is needed to improve the assessment of dense trabeculae.

Metabolic abnormalities caused by 3-acetylpyridine in the cerebral motor regions of rats: partial recovery by thyrotropin-releasing hormone.

Although 3-acetylpyridine (3-AP) induces several motor disturbances and it degenerates the olivocerebellar pathway, abnormalities caused by 3-AP in cerebral motor regions remain to be elucidated. Here we investigated the metabolic changes caused by 3-AP (75 mg/kg, i.p.) on local cerebral glucose utilization (LCGU) in various brain regions. The effects of anti-ataxic agents, thyrotropin-releasing hormone (TRH) (10 mg/kg, i.p.) and its mimetic agent taltirelin hydrate (1 mg/kg, i.p.), on the 3-AP-induced change in LCGU were also investigated. The LCGU in the nuclei of the basal ganglia, thalamus, limbic structures and brainstem of 3-AP-treated rats was significantly lower than that of naive animals. However 3-AP increased the LCGU of the cerebellar nuclei. TRH restored depressed LCGU in the substantia nigra and ventral tegmental area. TRH tended to restore the lowered LCGU in several nuclei of 3-AP-treated rats. Moreover, taltirelin further increased the LCGU in the cerebellar nuclei. These results suggest that the motor disturbance of the 3-AP-treated rats may be due to not only degeneration of the olivocerebellar pathway but also dysfunction of the several areas that play a role in motor coordination. Moreover, the anti-ataxic action by TRH could result from metabolic restoration of the multiple motor-coordination-related areas.

[A comparison between three methods to investigate falls among the elderly living in the community].

PURPOSE: Interview survey is the often selected method to investigate falls among the elderly. However, the reliability of data obtained by that method has not be confirmed. The aim of this study is to clarify the number of intervals of recollection that should be specified to ensure accurate data when we investigate falls by using interview surveys among the elderly. METHODS: We carried out a total health survey among the elderly in Shigenobu town, and reported about the frequency of falls and its relating factors. Sex, age, walking ability over a distance of 1 km and hospitalization experience for past 1 year were related to falls significantly. In this study, we set 3 groups matching for the above factors in addition to history of falls during the preceding a year, and for one year we asked about falls every month for the first group (116 participants), every 3 months for the second group (116 participants) respectively. The third group (118 participants) was asked once at the end of this study. These three groups totaled 350 persons who were participants of physical fitness measurements that we carried out in Shigenobu town. The existence of falls was investigated by mail, and the nonrespondents were questioned by direct telephone calls. RESULTS: For reasons such as death, moving out, hospitalization and answer denial, persons whose answer was not obtained were excluded from the analysis. Finally, 87.1% in the 1st group, and 89.7% in the 2nd group and 96.6% in the 3rd group were analyzed. Statistically significant difference for the above factors related to falls did not exist among the 3 groups. Annual incidence for falls tended to be more frequent in the 1st group that in the 3rd group (20.5% in the 1st group, 15.9% in the 2nd group and 6.4% in the 3rd group) in males. In females, such a difference was not observed. (26.3% in the 1st group, 18.3% in the 2nd group and 20.9% in the 3rd group). CONCLUSIONS: There were no significant differences for the factors related to falls in 3 groups, therefore, the groups had almost similar backgrounds. In men, the difference of annual reported incidence between the 1st group and the 3rd group may be due to differences in the method of recollection.

[Clinical training and home therapy in our clinic].

Since 1986, we have practiced clinical training. In this training, the physician trainee acts as the head of our clinic, managing programs including the medical teams for outpatient clinics and home therapy. Our home therapy started in 1975 and successive resident trainees have held conferences, cooperation between hospitals and clinics between clinics, and also held home patient and family parties. In our clinical training, therefore, the resident can effectively gain generalized clinical abilities.

Starting position of movement and perception of angle of trunk flexion while standing with eyes closed.

The present study attempted to investigate the effect of position on the perception of angle of trunk flexion while standing. For this purpose, the range effect was factored out by setting the constant target angle at 10 degrees, with varied starting positions of trunk flexion. We found that subjects underestimated angle of trunk flexion when the starting position was close to a quiet standing posture, overestimated when close to maximum trunk flexion, and correctly perceived it when at the middle position. Less perceptual distortion was observed at the positions close to maximum trunk flexion in the present study than in our previous one, in which various target angles of trunk flexion were reproduced from a quiet standing posture. The reduced distortion in the present study was believed to have resulted from factoring out the range effect. The flexion angle of the hip joint changed in tandem with that of the trunk, while very little movement was observed in the ankle, knee, and neck joints. Judging from the changing pattern of hip-joint angle, the muscle activity of the erector spinae and biceps femoris increased gradually to 90 degrees trunk flexion. In contrast, the actual increment of muscle activity reached zero or a minimum value at the middle angles as the angle of trunk flexion increased. It was assumed that the abrupt change in kinesthetic information associated with muscle activity exerted a great influence on the perception of trunk flexion.

Perceptibility of body position in anteroposterior direction while standing with eyes closed.

The perception of body position in the anteroposterior direction was investigated by evaluating the reproducibility of the position from a quiet standing posture to forward or backward leaning posture with eyes closed. The subjects were 10 healthy male undergraduates, aged 20 to 28 years. The standing position was represented by the pressure center of the foot, which was shown by the relative distance (%) from the heel to the length of the foot. The reference positions of the pressure center of the foot were set at 10% increments from 20 to 80% of the length of the foot. The subjects attempted to reproduce each reference position 10 times, and the absolute and constant errors of the reproduced position were analyzed. The absolute errors at reference positions of 30 to 60% were distinctly larger than those at the other reference positions. This indicated that the perception of standing positions from 30 to 60% was less accurate. The constant errors at the reference positions of 40 to 60% were significantly positive, which meant that the reproduced position was located farther forward than the reference position.

Production of ex-germfree rabbits for establishment of specific pathogen-free (SPF) colonies.

Nine groups of ex-germfree (GF) rabbits were produced by inoculation of hysterectomy-derived GF rabbits with various combinations of cecal bacteria isolated from conventional (CV) rabbits in order to establish a barrier-sustained colony. Six strains of Bacteroides and two strains of Streptococcus isolated from CV rabbits (2 to 3 weeks old) were used for pretreatment. The mortality of ex-GF rabbits inoculated with the anaerobic growth (CF) on EG or SM10 plates inoculated with a 10(-5) dilution of cecal contents was 71.4 to 94.4% when given without pretreatment. All ex-GF rabbits pretreated with Bacteroides alone survived, but the mortality of ex-GF rabbits inoculated with Bacteroides plus Streptococcus strains as pretreatment was 20 and 45.4%. The mortality of ex-GF rabbits inoculated with only Bacteroides was 43%. All ex-GF rabbits inoculated with Bacteroides plus anaerobic growth (CF), cecal suspension of ex-GF mice which had been inoculated with cecal suspensions of CV rabbits (MF) or chloroform-treated cecal suspension (CHF) survived, but CHF inoculated ex-GF rabbits were in an unhealthy condition with slight diarrhoea. These data indicate that inoculation with Bacteroides strains as pretreatment plus CF or MF was required to convert GF rabbits to the normal state.

Establishment of specific pathogen-free rabbit colonies with limited-flora rabbits associated with conventional rabbit flora, and monitoring of their cecal flora.

In the present study we attempted to establish specific-pathogen-free (SPF) rabbit breeding colonies with two groups of limited-flora (LF) rabbits, both ex-germfree rabbits, and their offspring. Two groups of LF rabbits associated with cecal flora of conventional (CV) rabbits produced in a previous study [Exp. Animals, submitted], were transferred to individual barrier rooms and some of the LF rabbits were accommodated in isolators to maintain the basic flora for SPF rabbits. The composition of the cecal flora of LF rabbits was stable for a long period; bacteroides remained predominant and clostridia dominant. From the SPF rabbits, different types of bacteria, e.g., enterobacteriaceae and streptococci, which could not be isolated in the isolator were detected at a low population level at an early stage in the establishment of the SPF colonies, but the basic composition of the cecal flora was mainly bacteroidaceae and clostridia and did not change over a long period, and the floral composition became similar to that of CV rabbits. The fertility and weaning rates of the SPF rabbits were satisfactory for a SPF rabbit colony. In addition, these SPF colonies were free of more than one year rabbit-specific pathogens.