Fibrin glue is a candidate scaffold for long-term therapeutic protein expression in spontaneously differentiated adipocytes in vitro.

Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional protein translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy.

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.

Disturbed apolipoprotein A-I-containing lipoproteins in fish-eye disease are improved by the lecithin:cholesterol acyltransferase produced by gene-transduced adipocytes in vitro.

We report the in vitro efficacy of recombinant LCAT produced by lcat gene-transduced proliferative adipocytes (ccdPA/lcat), which has been developed for enzyme replacement therapy. ApoA-I-specific immunodetection in combination with 1D and 2D gel electrophoreses showed that the disturbed high-density lipoprotein subpopulation profile was clearly ameliorated by the in vitro incubation with ccdPA/lcat-derived recombinant LCAT. Thus, these results using ccdPA/lcat strongly suggest the cell implantation could contribute the enzyme replacement for the patients with LCAT deficiency.

Stratification of disease progression in a broad spectrum of degenerative cerebellar ataxias with a clustering method using MRI-based atrophy rates of brain structures.

BACKGROUND: The rate of disease progression differs among patients with degenerative cerebellar ataxia. The uncertain natural course in individual patients hinders clinical trials of promising treatments. In this study, we analyzed atrophy changes in brain structures with cluster analysis to find sub-groups of patients with homogenous symptom progression in a broad spectrum of degenerative cerebellar ataxias. METHODS: We examined 48 patients including 21 cases of spinocerebellar ataxia (SCA), 17 cases of the cerebellar type of multiple system atrophy (MSA-C), and 10 cases of cortical cerebellar ataxia (CCA). In all patients, at least two sets of evaluations including magnetic resonance imaging (MRI) and the International Cooperative Ataxia Rating Scale (ICARS) scoring were performed. The median number (min-max) of follow-up studies in each patient was three (2-6), and the mean follow-up period was 3.1 ± 1.6 years. The area of the corpus callosum on midsagittal images and the cerebellar volume were measured using MRI, and these values were divided by the cranial antero-posterior diameter of each patient to correct for individual head size differences as an area index (Adx) and a volume index (Vdx), respectively. The annual changes in Adx, Vdx, and ICARS score were calculated in each patient, and atrophy patterns in patients were categorized with cluster analysis. RESULTS: The annual atrophy rates for the corpus callosum (Adx) and cerebellum (Vdx) and symptom progression differed significantly by subtype of cerebellar ataxia (p = 0.026, 0.019, and 0.021, respectively). However, neither the annual atrophy rate of Adx nor Vdx was significantly correlated with the annual increase in the ICARS score. When the patients were categorized into three clusters based on the annual changes in Adx and Vdx, the annual increase in the ICARS score was significantly different among clusters (2.9 ± 1.7/year in Cluster 1, 4.8 ± 3.2/year in Cluster 2, and 8.7 ± 6.1/year in Cluster 3; p = 0.014). CONCLUSIONS: The annual increase in the ICARS score can be stratified by cluster analysis based on the atrophy rates of the corpus callosum and cerebellum. Further studies are warranted to explore whether these simple MRI methods could be used for random allocation of a broad spectrum of patients with degenerative cerebellar ataxia in clinical trials.

MRI-based cerebellar volume measurements correlate with the International Cooperative Ataxia Rating Scale score in patients with spinocerebellar degeneration or multiple system atrophy.

BACKGROUND: Progression of clinical symptoms and cerebellar atrophy may vary among subtypes of spinocerebellar degeneration and multiple system atrophy. The aim of this cross-sectional study was to demonstrate the relationship between the International Cooperative Ataxia Rating Scale (ICARS) score and cerebellar volume derived from magnetic resonance imaging (MRI) in a broad spectrum of Japanese patients with cerebellar ataxia. METHODS: A total of 86 patients with cerebellar ataxia (18 with cortical cerebellar atrophy, 34 with spinocerebellar ataxia, and 34 with multiple system atrophy) and 30 healthy subjects were studied. MRI-based cerebellar volume measurements were performed in all subjects using T1-weighted images acquired with a 1.5-T MRI scanner. The cerebellar volume/cranial anteroposterior (AP) diameter was used for statistical analysis. RESULTS: Stepwise multiple regression analyses demonstrated that cerebellar volume/cranial AP diameter and midbrain AP/cranial AP diameter were significantly associated with the total score and domain I sub-score of ICARS. We found no interactions between these two anatomical factors in the ICARS total and domain I sub-scores. The main effects of these two predictors were statistically significant both in total and domain I sub-scores (p = 0.001 and 0.022, respectively). CONCLUSIONS: Cerebellar volume and midbrain AP diameter normalized to the cranial AP diameter were significantly correlated with the ICARS total and domain I sub-scores. Further longitudinal studies are warranted to explore the role of these MRI biomarkers for predicting disease progression.

[Life-threatening airway obstruction accompanied by vocal cord paralysis due to indwelling nasogastric tube in malnourished elderly patients: a report of four cases].

We report 4 cases of elderly patients with abrupt onset of serious airway obstruction that is presumed to be due to indwelling nasogastric tube. 2 cases are patients of cerebral infarction and 2 cases are patients of Parkinson disease. The average number of days until NGTS is 17.8 days. In all cases, fiber-optic examination revealed complete loss of adduction in both vocal cords. Infection in the posterior cricoid region caused by ulcerative lesions at the upper end of the esophagus has been implicated as a pathophysiological mechanism of this syndrome, but it was not possible to confirm in the 4 cases. Because it is difficult to exactly diagnose with NGTS in clinical practice, there is a need to consider the inducing factor and response. Body mass index is very low in each of the 4 cases, ranging from 14.2 to 18.0, implying a severely malnourished or immunocompromised state, and may represent a high risk factor for this syndrome. Whenever this life-threatening syndrome is suspected, direct vocal cord examination and removal of the tube are recommended. In addition, the clinicians should not hesitate about doing intubation or tracheotomy in emergency.

Design and Semisynthesis of Photoactive Myoglobin Bearing Ruthenium Tris(2,2'-bipyridine) Using Cofactor-Reconstitution.

A new strategy for semisynthesis of a photoactivatable redox protein is described. Three protohemin molecules with ruthenium tris(2,2'-bipyridine) attached by different spacers were synthesized. The Ru(bpy)(3)-protohemins were incorporated into the heme crevice of apomyoglobin (apo-Mb) to yield semisynthetic Mbs carrying Ru(bpy)(3) as a photosensitizer (Ru(bpy)(3)-Mb). The photoactivation properties and the reaction mechanisms of Ru(bpy)(3)-Mbs were investigated by steady-state photoirradiation and laser flash photolysis. The photoactivation of Ru(bpy)(3)-Mbs was spectrophotometrically demonstrated by comparison with an intermolecular control, namely an equimolar mixture of Ru(bpy)(3) and native Mb. The spacer structure considerably influenced net activation efficiency over a wide pH range as measured by steady-state visible light irradiation and quantum yield. Laser flash photolysis yielded the rate of the photoinduced electron transfer (ET) from the lifetime of the excited Ru(bpy)(3) (k(et) = 4.4 x 10(7) s(-)(1) for Mb(1b) and k(et) = 3.7 x 10(7) s(-)(1) for Mb(1c)) and the back ET rate (k(back) = (2.0-3.7) x 10(7) s(-)(1) for Mb(1b) and k(back) = (1.4-2.4) x 10(7) s(-)(1) for Mb(1c)) from the decay of the transient absorption. These data consistently explained the results of the net photoreaction as follows. (i) The intermolecular control system was less photoactivated because little ET occurred from the excited state of Ru(bpy)(3) to Mb. (ii) The short lifetime of the charge-separated state after photoinduced ET greatly decreased the photoactivation efficiency of Ru(bpy)(3)-Mb with the shortest spacer. (iii) The photochemical and photophysical data of the other two Ru(bpy)(3)-Mb derivatives (the net photoreaction, quantum yield, and ET/back ET rates) were essentially identical, indicating that flexible spacers consisting of oxyethylene units do not rigidly fix the distance between Ru(bpy)(3) and the heme center of Mb. In addition, Ru(bpy)(3)-Mbs were highly photoactivated under aerobic conditions in a manner similar to that under anaerobic conditions, although O(2) usually quenches the photoexcited state of Ru(bpy)(3). This was probably due to the accelerated intramolecular ET from Ru(bpy)(3) to heme, not to O(2) in Ru(bpy)(3)-Mbs. We therefore showed that visible light affects the content of O(2)-bound Mb even in air.

Fibrin glue increases the cell survival and the transduced gene product secretion of the ceiling culture-derived adipocytes transplanted in mice.

The development of clinically applicable scaffolds is important for the application of cell transplantation in various human diseases. The aims of this study are to evaluate fibrin glue in a novel protein replacement therapy using proliferative adipocytes and to develop a mouse model system to monitor the delivery of the transgene product into the blood and the fate of the transduced cells after transplantation. Proliferative adipocytes from mouse adipose tissue were transduced by a retroviral vector harboring the human lecithin-cholesterol acyltransferase (lcat) gene, and were subcutaneously transplanted into mice combined with fibrin glue. The lcat gene transduction efficiency and the subsequent secretion of the product in mouse adipocytes were enhanced using a protamine concentration of 500 µg/ml. Adipogenesis induction did not significantly affect the lcat gene-transduced cell survival after transplantation. Immunohistochemistry showed the ectopic enzyme production to persist for 28 days in the subcutaneously transplanted gene- transduced adipocytes. The increased viability of transplanted cells with fibrin glue was accompanied with the decrease in apoptotic cell death. The immunodetectable serum LCAT levels in mice implanted with the fibrin glue were comparable with those observed in mice implanted with Matrigel, indicating that the transplanted lcat gene-transduced adipocytes survived and functioned in the transplanted spaces with fibrin glue as well as with Matrigel for 28 days. Thus, this in vivo system using fibrin is expected to serve as a good model to further improve the transplanted cell/scaffold conditions for the stable and durable cell-based replacement of defective proteins in patients with LCAT deficiency.