Optimization of cell-wall skeleton derived from Mycobacterium bovis BCG Tokyo 172 (SMP-105) emulsion in delayed-type hypersensitivity and antitumor models.

Cell-wall skeleton prepared from Mycobacterium bovis BCG (BCG-CWS) is known as a potent adjuvant and has been shown to possess antitumor activity in many non-clinical and clinical studies. As there are no approved BCG-CWS formulations for cancer therapy, we investigated the potential for cancer immunotherapy of SMP-105, our originally produced BCG-CWS. For optimizing SMP-105 emulsion, we compared the effects of drakeoland squalane-based SMP-105 emulsions on IFN-γ production in rats and evaluated their ability to induce skin reaction in guinea pigs. Both emulsions had the same activity in both experiments. We selected squalane as base material and produced two types of squalane-based formulations (vialed emulsion and pumped emulsion) that can easily be prepared as oil-in-water emulsions. Although the vialed emulsion showed the same pattern of distribution as a usual homogenized emulsion, the pumped emulsion showed more uniform distribution than the other two emulsions. Whereas both emulsions enhanced strong delayed type hypersensitivity (DTH) reaction in a mouse model, the pumped emulsion induced slightly smaller edema. Data on oil droplet size distribution suggest that few micrometer oil droplet size might be appropriate for oil-in-water microemulsion of SMP-105. The antitumor potency of SMP-105 emulsion was stronger than that of some of the launched toll-like receptor (TLR) agonists (Aldara cream, Picibanil, and Immunobladder). Aldara and Picibanil showed limited antitumor effectiveness, while Immunobladder had almost the same effect as SMP-105 at the highest dose, but needed about 10 times the amount of SMP-105. These findings first indicate that SMP-105 has great potential in cancer immunotherapy.

Childhood T-cell acute lymphoblastic leukemia with four distinct immunophenotypes representing different stages of T-cell development.

The authors report on a 14-year-old boy who developed T-cell acute lymphoblastic leukemia (FAB:L1) displaying 4 immunophenotypically distinct leukemic cell populations by 3-color immunofluorescence staining. Cytogenetic analysis at diagnosis showed 46,XY,add(4)(p16)[12]/46,XY[2]. A single rearrangement of the T-cell antigen receptor beta- and gamma-chain genes in these cells indicated monoclonality of the leukemic cells. These findings suggest that leukemic blast cells of monoclonal origin in this case were divided into 4 immunophenotypic populations, representing various stages of differentiation.

[A case of Miller-Dieker syndrome associated with satellite on chromosome 17p].

In this report, we describe a one-year-old girl of the Miller-Dieker syndrome(MDS) with lissencephaly, seizures, microcephaly and mental disorders. Cytogenetic studies of this patient confirmed the presence of a 46,XX, 17ps+ chromosome karyotype, but it could not find the microdeletion of 17p13.3. Fluorescence in situ hybridization(FISH) studies confirmed a terminal deletion in the patient using the LIS1 gene probe which mapped to 17p13.3. Further it was also found the satellite on 17p13(17ps) in the patient who was rare associated with MDS. These findings suggest that FISH analysis may be useful method to detect microdeletion of LIS1 gene as 17-specific probe in the investigation of MDS patients.

[A case of 9p-syndrome identified chromosome 20 on chromosome 9p by M-FISH].

In this report, we describe one-year-old girl diagnosed with 9p-syndrome. Cytogenetic studies of this patient confirmed a karyotype of 46,XX,add(9) (p24) chromosome, but could not find the additional fragment on 9p22 in one allele. Fluorescence in situ hybridization (FISH) studies could not confirm the fragment in the patient using the LIS1 gene probe which mapped to 9p22. The more recently developed M-FISH method clearly showed that the additional fragment was 20p in this patient. These findings suggest that M-FISH analysis may be a useful method for identifying unknown additional and rearranged chromosomes.

Cell cycle arrest enhances the in vitro cellular toxicity of the truncated Machado-Joseph disease gene product with an expanded polyglutamine stretch.

Machado-Joseph disease (MJD) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine stretch in the MJD gene-encoded protein, ataxin-3. Using a series of deletion constructs expressing ataxin-3 fragments with expanded polyglutamine stretches, we observed aggregate formation and cell death in cultured BHK-21 cells. The cytotoxic effect of N-terminal-truncated ataxin-3 with the expanded polyglutamine tract was enhanced under serum starvation culture, in which cells were arrested in the G(0)/G(1)phase. Coexpression of p21 (waf1/cip1/sdi1), a cyclin-Cdk inhibitor that induced cell cycle arrest in the G(1)phase, also increased the cell death susceptibility produced by the mutant ataxin-3 fragment in BHK-21 cells. The elevated susceptibility to cell death in the G(0)/G(1)phase was confirmed in nerve growth factor-treated, postmitotic neuronal PC12 cells compared with undifferentiated proliferating PC12 cells. These results strongly suggest that the cellular toxicity of truncated ataxin-3 with an expanded polyglutamine stretch is enhanced by cell cycle arrest in the G(0)/G(1)phase. Mutant ataxin-3 may confer a higher susceptibility to cell death on cells in the G(0)/G(1)phase.

BDNF atelocollagen mini-pellet accelerates facial nerve regeneration.

We investigated the effect of BDNF mini-pellet on the GAP-43 mRNA expression and functional status of facial nerve in a rat model of facial nerve transection and immediate repair. The facial function started to recover at 17 days in the placebo group and 14 days in the BDNF group. BDNF group had shorter period of increased GAP-43 mRNA expression than the placebo group. Topically applied BDNF may accelerate the facial nerve regeneration.

Influence of myopic disc shape on the diagnostic precision of the Heidelberg Retina Tomograph.

PURPOSE: To investigate the diagnostic capability of a glaucoma diagnostic classification program for the Heidelberg Retina Tomograph (HRT) in eyes with myopic disc shapes. METHODS: Sixty-six normal subjects (66 eyes) and 78 open-angle glaucoma patients (78 eyes) were enrolled. The eyes were divided into two groups; those eyes with myopic and those with nonmyopic disc shapes. The classification was based on clinical judgment made after the examination of stereophotographs of the discs without considering the refractive errors. The agreement between the classification program and the clinical diagnosis was evaluated for sensitivity, specificity, and diagnostic precision. The influence of the disc shape on the HRT topographic parameters was evaluated. RESULTS: The sensitivity, specificity, and diagnostic precision of the HRT were 83%, 95%, and 89% in eyes with nonmyopic disc shapes, but 71%, 96%, and 83% in those with myopic disc shapes. Rim volume, height variation contour, mean retinal fiber nerve layer (RNFL) thickness, and RNFL cross-section area were significantly larger in eyes with myopic disc shapes than in eyes with nonmyopic discs, regardless of the clinical diagnosis. CONCLUSIONS: The classification program should be modified to take into account the myopic disc shape in order to improve its capability to make more accurate diagnosis of glaucoma possible.

[Influence of myopic disc shape in a classification program of the Heidelberg retina tomograph].

PURPOSE: We investigated the influence of myopic disc shape on the diagnostic capability of a glaucoma diagnostic software (classification program) of the Heidelberg retina tomograph (HRT). SUBJECTS: 66 eyes of 66 normal subjects and 78 eyes of 78 patients with open-angle glaucoma were studied. The criterion of glaucoma was a visual field defect appearing between Aulhorn classification stage II and stage V regardless of the maximum intraocular pressure value. METHODS: The subjects were divided into eyes with a myopic disc and those with a non-myopic disc on the basis of stereo disc fundus photographs without considering the refractive errors. Agreement between the classification program and the clinical diagnosis was evaluated by the calculation of sensitivity, specificity, and diagnostic precision, and the influence of the disc shape on HRT disc shape parameters was also evaluated. RESULTS: Sensitivity, specificity, and diagnostic precision of the classification program were 83%, 95%, and 89% in the eyes with a non-myopic disc, and 71%, 96%, and 83% in the eyes with a myopic disc, respectively. Rim volume, height variation contour, mean RNFL (retinal nerve fiber layer) thickness, and RNFL cross section area were significantly larger in the eyes with a myopic disc than in those with a non-myopic disc regardless of the clinical diagnosis. CONCLUSION: The classification program should be modified to adjust to a myopia-like disc shape in order to improve the capability of the glaucoma predictive diagnosis.

Effects of oral brovincamine on visual field damage in patients with normal-tension glaucoma with low-normal intraocular pressure.

PURPOSE: To prospectively study the effect of oral brovincamine, a relatively selective cerebral vasodilator, on further deterioration of visual field in patients with normal-tension glaucoma (NTG) with low-normal intraocular pressure (IOP). METHODS: Fifty-two patients with NTG (average age 57.7 years) with an IOP that was consistently less than 15 mmHg were randomly assigned to receive oral brovincamine (20 mg three times daily) or to an untreated control group. The groups were prospectively followed for 2 years with visual field examinations every 4 months, using the 30-2 Humphrey perimeter program. Changes in mean deviation (MD), corrected pattern standard deviation (CPSD), and total deviation (TD) at 74 test points were analyzed using regression analysis with linear mixed model. Data from one eye without media opacity of each subject were analyzed. RESULTS: There were no differences between groups in age; sex distribution; refraction; blood pressure; baseline IOP; MD, CPSD, or TD at each point. Changes in MD (standard error [SE]) during the study period were -0.778 (0.178) and -0.071 (0.195) dB/year in the control and brovincamine groups, respectively; change in the control group was significantly more negative than in the brovincamine group. Change in CPSD (SE) was 0.032 (0.015) and 0.004 (0.016) dB/year in the control and brovincamine groups, respectively. Change in the control group was significantly positive, but the intergroup difference was not significant. Change in TD was significantly negative at six test points in the control group, whereas no points showed a significant trend in the brovincamine group; the intergroup difference was significant. The average IOP was 13.2 mmHg and 13.1 mmHg in the control and brovincamine groups, respectively, and there was no significant intergroup difference. CONCLUSION: Oral brovincamine may retard further visual field deterioration in patients with NTG who have low-normal IOP.

Distribution of nerve growth factor in cat brains following topical application of solution or Minipellet.

The distribution of nerve growth factor (NGF) was studied after topical application of NGF solution or NGF Minipellet into the caudate nucleus of cat brains. Each aliquot of NGF solution or a piece of NGF Minipellet contains 200 micrograms of mouse 2.5S beta-NGF. The concentrations of NGF in various areas of the brain were determined by enzyme-linked immunosolvent assay (ELISA). The tissue concentrations of NGF were very high at 6 h after injection of NGF solution (124.0 +/- 2.46 to 2144.0 +/- 16.03 ng g-1), but quickly decreased on day 1. The tissue concentrations of NGF were low at 6 h after implantation of NGF Minipellet, but were considerably increased on day 1 (11.22 +/- 3.36 to 72.04 +/- 20.45 ng g-1), and slowly decreased during the following 6 to 14 days. The present study demonstrates the temporal and spatial profiles of NGF distribution in the brain of middle-sized animals after topical application. Injection of NGF solution resulted in abrupt, but very transient, elevation of tissue NGF. Implantation of NGF Minipellet maintained the tissue NGF at biologically effective levels for 6 to 14 days.

[Effect of non-surgical ocular hypotensive therapy in normal-tension glaucoma].

We investigated the effects of the topical ocular hypotensives with or without half circumference argon laser trabeculoplasty (ALT) on intraocular pressure (IOP) of normal-tension glaucoma (NTG) eyes. Seventy-two NTG patients whose pretreatment mean IOP was > or = 15 mmHg at least in one eye were included and one randomly chosen eye from one patient were used for analysis. They were followed for at least 15 months after commencement of the therapy. The mean pretreatment IOP averaged 17.2 +/- 1.6 mmHg (mean +/- standard deviation, n = 72). All eyes were first treated with topical 2% carteolol. In eyes where IOP reduction was considered unsatisfactory, topical 1% pilocarpine or 0.04% dipivefrine was added. In eyes where IOP reduction was still unsatisfactory even with the two medications, half circumference ALT was performed. Mean IOP reduction was 1.5 mmHg in the eyes treated with topical medications alone and 2.5 mmHg in those treated by topical medications plus ALT. The mean outflow pressure reduction was 16 and 26%, respectively. In 40% of the all eyes, satisfactory IOP reduction was obtained by topical medications with or without additional ALT.

Effect of trabeculectomy on visual field performance in central 30 degrees field in progressive normal-tension glaucoma.

PURPOSE: The authors studied the effects of trabeculectomy on the time course of central 30 degrees visual field performances in progressive normal-tension glaucoma (NTG). METHODS: Patients with NTG who had clear ocular media and were adequate performers on the 30-2 program of Humphrey Perimeter were prospectively followed with periodic field testing. Trabeculectomy using an antimetabolite was indicated when the slope of a line fitted to the time course of the mean deviation (MD), MD slope, was significantly negative. The time courses of MD and mean of total deviations (mean TD) in four subfields, superior and inferior arcuate and superior and inferior cecocentral fields, were analyzed using the mixed linear model. RESULTS: In progressive NTG, 21 eyes of 21 cases with postoperative follow-up of 2 years or more, intraocular pressure averaged 16 mmHg, MD averaged -13.5 dB, and MD slope averaged -1.48 dB/year preoperatively; 2 years after surgery, they averaged 9.2 mmHg, -13.6 dB, and +0.13 dB/year, respectively. Trabeculectomy had a significant beneficial effect on the time course of MD and mean TD in the superior and inferior arcuate and superior cecocentral fields, which showed significant preoperative deterioration. The mean TD in the inferior cecocentral field showed no significant time change during the period studied. CONCLUSION: For patients with NTG in whom the MD slope is significantly negative, trabeculectomy may have beneficial and diffuse effects on further deterioration of the visual field.

Intraocular pressure and central visual field of normal tension glaucoma.

AIM: To study whether damage in the central 30 degrees field of normal tension glaucoma (NTG) is relatively heterogeneous or homogeneous with respect to intraocular pressure (IOP) related damage. METHODS: Using the results of Humphrey perimeter examinations, the central 30 degrees field was divided into four subfields; superior and inferior hemifields excluding the caecocentral field (30-2 program) and superior and inferior 10 degrees hemifields (10-2 program). In 103 NTG cases, the intraindividual bilateral difference in the mean of total deviations (mean TD) in the four subfields was analysed by multiple linear regression to correct the effects of factors other than IOP. Explanatory variables were the intraindividual bilateral difference in the mean of clinic IOP (IOPmean), that in the ratio of area of peripapillary atrophy corresponding to each subfield to disc area, and that in myopic refraction. RESULTS: The intraindividual bilateral difference in the mean TD was significantly and negatively correlated with that in IOPmean in three of the above four subfields (p < 0.005) and correlation tended to be negative (p = 0.07) in the superior 10 degrees hemifield. CONCLUSIONS: Diffuse IOP related damage was suggested in the central 30 degrees field of NTG; greater extent of the damage in the above four subfields was correlated with higher mean IOP. The present findings may have clinical implications.

Suppression of growth-associated phosphorylation of proteins of fibroblasts by collagen fibrils.

Collagen fibrils suppressed serum- or epidermal growth factor (EGF)-inducible DNA synthesis of human fibroblasts. The phosphorylation of cellular proteins upon these mitogenic stimulation was analyzed by two-dimensional polyacrylamide gel electrophoresis in order to reveal a possible interference of collagen fibrils with the cellular mitogenic signal transduction pathway coupled with the protein phosphorylation-dephosphorylation reaction. Spots of phosphorylated proteins numbered 192 on plain plastic which were reduced to 143 on collagen fibrils. More than half of them were matched between the two substrates, most of which were much more weakly phosphorylated on collagen fibrils. EGF stimulated the phosphorylation of these proteins of cells on plastic. Among them a protein with an approximate molecular weight of 27K and an isoelectric point of 5.3 was early and highly responsive to EGF, phosphorylation of which seemed to be catalyzed mainly by protein kinase C and tyrosine kinase. Collagen fibrils significantly suppressed this phosphorylation. The present study demonstrates that collagen fibrils modulate the growth-associated protein phosphorylation of cells, which seems to lead to the suppression of DNA synthesis.

Color vision deficit in normal-tension glaucoma eyes.

Color vision deficit in 26 eyes with normal-tension glaucoma (NTG) is compared with that in 21 eyes with primary open angle glaucoma (POAG) using the color perimetry method developed by Iijima et al. Subjects had visual field defects confined to either the upper or the lower hemifield as determined by conventional white-on-white perimetry, and the stage of disease was relatively early with an average mean deviation (MD) of -7 dB. Except for intraocular pressure, there were no significant differences between NTG and POAG subjects in age, refraction, MD, and mean total deviation for spared and damaged hemifield. In the spared hemifield, the incidence of blue-yellow abnormality was significantly different between the two diseases (P = 0.01); in only 11% of the present NTG eyes versus 52% of the POAG eyes in the present study. In the damaged hemifield, however, the blue-yellow abnormality was found in about 75% of the eyes, whether NTG or POAG. This finding may further shed light on the pathogenesis of open angle glaucoma.

Influence of myopic refraction on visual field defects in normal tension and primary open angle glaucoma.

It is known that the prevalence of normal tension glaucoma (NTG) or primary open angle glaucoma (POAG) is higher in the myopic population and that the prevalence of myopia and NTG is higher in Japan than in western countries. To evaluate the influence of myopic refractive error in the central 10 degrees of the visual field in NTG eyes, the data obtained from NTG eyes with the Humphrey Visual Field Analyzer were analyzed in comparison with data from POAG eyes. One hundred and twenty eyes of 86 NTG patients and 197 eyes of 138 POAG patients whose ages were 65 years or younger were included in this study. All of them had clear ocular media, refractive power less than +1.0 diopter and no myopic degeneration in the fundus. Multiple linear regression analysis was performed with the dependent variable of the deviation of the measured threshold value from the normal reference value (TD; STATPAC 2), and independent variables of myopic refractive power and the mean deviation (MD; STATPAC 2) as an index of the disease stage at each test point of the Humphrey 10-2 program and at the most central 12 test points of the 30-2 program. Myopic power was found to have a significant positive correlation with the depression in the lower cecocentral area in both NTG and POAG eyes, while only in NTG eyes it had a significant negative correlation with the depression in the upper arcuate area extending just nasal to the fixation point.

Visual field damage proximal to fixation in normal- and high-tension glaucoma eyes.

To compare in more detail the central visual field damage in normal-tension glaucoma (NTG) and high-tension glaucoma (HTG) eyes, a pointwise inner-group comparison of the data obtained with the Humphrey 10-2 was carried out in 68 NTG cases with maximum intraocular pressure (IOP) of < or = 21 mmHg and 62 HTG cases with maximum IOP of > or = 25 mmHg. All eyes had the usual field defects, with a mean deviation of > or = -15 dB (MD, STATPAC). Age, refraction and MD were matched between the two groups. Total deviation (TD, STATPAC), the difference between the measured threshold and the age-corrected normal reference at each test point of the 10-2 program, was used for pointwise inter-group comparisons. The data were analyzed with Wilcoxon rank-sum test and logistic discriminant analysis. The latter method was also applied to the data of the 30-2 program to confirm the results obtained with the 10-2 program. The comparisons using the different methods and programs gave consistent results. For a given amount of overall visual field damage in the two types of glaucoma, a superior arcuate area extending down to the horizontal meridian just nasal to the fixation point was significantly more depressed and an area inferior to the horizontal meridian and inferior temporal to the fixation point significantly less depressed in NTG eyes. HTG eyes were significantly more diffusely damaged in the central 10-degree visual field. NTG and HTG may have unique patterns of central visual field damage. These differences may implications in following NTG patients and studying the pathogenesis of this glaucoma.

[Influence of refraction on the visual field defects in normal tension glaucoma and primary open angle glaucoma eyes].

The prevalence of normal tension glaucoma (NTG) or primary open angle glaucoma (POAG) is higher in myopic populations and the prevalence of myopia and NTG is relatively high in the Japanese. To evaluate the influence of myopic refractive error on the central visual field defect in NTG eyes, the data obtained from NTG eyes with the Humphrey Visual Field Analyser were analysed in comparison with those from POAG eyes. One hundred and twenty NTG eyes and one hundred and ninety-seven POAG eyes were included. All of them had clear ocular media, but no myopic degeneration in the fundus. Multiple linear regression analysis was performed with a dependent variable of the deviation of the measured threshold value from normal reference value (total deviation, STATPAC) and independent variables of refractive error and mean deviation (STATPAC). Myopic power was found to be positively correlated with the depression in the lower papillo-macular area in both NTG and POAG eyes, and it was negatively correlated with the depression in an upper arcuate area extending just nasal to the fixation only in NTG eyes.