Human adult endothelial cell growth in culture

The purpose of the present study was to culture human adult endothelial cells (HAECs) on a long-term basis in the laboratory. Previous inability to accomplish this has been the major impediment to the in vitro study of endothelialization of prosthetic grafts with human cells, a problem of significant clinical relevance. We have been successful in developing a technique that allows HAECs from human adult arteries, veins, and capillaries to proliferate vigorously in culture for up to 80 population doublings. HAECs are grown on a gelatin surface (medium 199 containing 20% fetal calf serum). Heparin and endothelial cell growth factor (ECGF) are required for optimal growth. With this technique, which will be described in detail, over 10(23) HAECs can be produced from each 1 cm2 of vascular tissue. This makes large numbers of HAECs available for high-density seeding on prosthetic grafts prior to implantation. It also permits for the first time with human cells the in vitro study of prosthetic grafts--HAEC interactions and the factors that enhance optimal growth and adherence to prosthetic materials. It is hoped that identification of the factors promoting graft endothelialization in combination with high-density seeding will lower graft thrombogenicity and therefore result in greater graft longevity than has been possible heretofore.     

Human intervertebral disc cell culture for disc disorders

Repair of degenerated intervertebral discs by engineered tissue is a clinical challenge in spinal surgery. Prerequisites are cultivation of intervertebral disc cells and determination of their biologic properties. The influence of disc damage in different spinal disorders on the outcome of disc cell cultures has not been discussed previously. This study showed the feasibility of cultivation of cells from damaged human intervertebral discs and the dependence of cellular culture properties on the underlying disc disorder. Human intervertebral disc cells were isolated from disc tissue obtained during surgical procedures for scoliosis, osteochondrosis, and disc herniation. After proliferation in monolayer culture, cells were embedded in a mixed matrix composed of fibrin and hyaluronic acid. Deoxyribonucleic acid content, hydroxyproline content, and proteoglycan synthesis were determined on Days 7, 14, and 21. In a three-dimensional environment only cells obtained from scoliotic and osteochondrotic discs showed significant deoxyribonucleic acid and proteoglycan synthesis. However, hydroxyproline content increased only in cells from scoliotic discs. The results of this study show that the formation of extracellular matrix components under three-dimensional culture conditions is dependent on the nature of intervertebral disc damage of the tissue processed.     

Human pregnancy after transfer of intact frozen-thawed embryos

Since the birth of the first baby as a result of in vitro fertilisation (IVF) in 1978, many clinics around the world have achieved pregnancies and births for their patients by using IVF and gamete intrafallopian transfer procedures. With the storage of excess embryos, multiple laparoscopies can be avoided; this has favoured the development of better cryopreservation techniques. In our clinic 8-cell human embryos are frozen in a 1.5M dimethyl sulphoxide solution as cryoprotectant using the slow freeze-thaw method. Sixteen thawed embryos were replaced in 8 patients, resulting in 1 pregnancy. Of the thawed embryos 51.6% survived the freezing process in that they had 50% or more of the original number of blastomeres and also the zona pellucida intact.     

Efficacy of nonviral gene transfer in the canine brain

OBJECT: The purpose of this study was to evaluate the gene transfer capability and tolerability of plasmid DNA/polyethylenimine (PEI) complexes in comparison with adenovirus and naked plasmid DNA in the canine brain. METHODS: Plasmid or adenoviral vectors encoding firefly luciferase were injected directly into the cerebral parenchyma of five adult dogs at varying doses and volumes. Serial physical and neurological examinations, as well as blood and cerebrospinal fluid (CSF) analyses, were conducted before and after the surgery for 3 days. Three days after gene delivery, a luciferase activity assay and immunofluorescence analysis were used to test the brain tissue for gene expression. RESULTS: Injection into the brain parenchyma resulted in gene transfer throughout the cerebrum with every vector tested. Luciferase expression was highest when adenovirus vectors were used. Injection of plasmid DNA/PEI complexes and naked DNA resulted in similar levels of luciferase expression, which were on average 0.5 to 1.5% of the expression achieved with adenovirus vectors. Immunofluorescent microscopy analysis revealed that plasmid DNA/PEI complexes transduced mainly neurons, whereas adenovirus transduced mainly astrocytes. No significant acute side effects or neurological complications were observed in any of the dogs. Mononuclear cell counts significantly increased in the CSF after adenovirus injection and modestly increased after injection of plasmid DNA/PEI complexes, suggesting that a mild, acute inflammatory response occurred in the central nervous system (CNS). CONCLUSIONS: Compared with rodent models that are limited by very small brains, the dog is an excellent preclinical model in which to assess the distribution and safety of emerging gene transfer technologies. In this study, short-term gene transfer was evaluated as a prelude to long-term expression and safety studies. The authors conclude that the viral and nonviral vectors tested were well tolerated and effective at mediating gene transfer throughout a large portion of the canine brain. The nonviral plasmid vectors were less effective than adenovirus, yet they still achieved appreciable gene expression levels. Due to reduced gene transfer efficiency relative to viral vectors, nonviral vectors may be most useful when the expressed protein is secreted or exerts a bystander effect. Nonviral vectors offer an alternative means to genetically modify cells within the CNS of large mammals.     

Analysis of mixed lymphocyte-tumor culture in patients with malignant brain tumor

To ascertain whether tumor-specific immune response occurs in patients with malignant brain tumors, lymphocyte blastogenetic responses to tumor cells were examined in 18 patients prior to operation and other treatment. Among 12 patients with malignant glioma, the peripheral blood lymphocytes (PBL's) showed a positive blastogenetic response to their own glioma cells in seven (58.3%), whereas the tumor-infiltrating lymphocytes (TIL's) showed a positive response in only three (25%). In four (66.7%) of six patients with metastatic brain tumors, however, both the PBL's and TIL's showed a positive blastogenetic response to their own tumor cells. In these four patients, this lymphocyte blastogenetic response to tumor cells were at a much lower level compared with phytohemagglutinin P or allogeneic lymphocyte stimulation. Furthermore, these responses were increased when the cells were cultured with interferon-gamma (500 U). Other lymphokines had no effect on the response. This method appears to be useful in identifying the tumor-specific immune response in patients with malignant brain tumor.     

Autologous mixed lymphocyte tumor cell culture in patients with renal cell carcinoma

Cell-mediated immunity was studied by measurement of lymphocyte response to autologous tumor cells in 19 surgically treated patients with histologically proved (mixed lymphocyte tumor cell culture) renal cell carcinoma. Tumor stage was low in 9 patients and high in 10, while grade was low in 11 and high in 8. Of 8 patients in whom a positive lymphocyte response was detected 6 had high and 2 had low stage tumors (p less than 0.05), while the grade of disease was low in 7 and high in 1 (p less than 0.05). Furthermore, the more advanced and undifferentiated the tumor the more significant the decrease in lymphocyte response (p less than 0.05). Lymphocyte response was positive in 5 of 8 patients with low stage and low grade tumors but negative in 7 with high stage and high grade disease. However, no correlation between the lymphocyte response and the degree of microscopic lymphocytic infiltration in and around the tumor was found. This study confirms that the specific immunological defense mechanism of patients with renal cell carcinoma against the tumors remains well at an earlier stage of tumor development, especially in cases with well differentiated malignancy, and showed attenuation in parallel with pathological spread or in poorly differentiated tumors.     

The characterization of tumor apoptosis after experimental radiosurgery

We sought to evaluate whether radiosurgery induces apoptosis in an experimental glioma model and to elucidate the time course of this radiobiologic phenomenon. Fischer 344 rats harboring established intracranial 9L gliosarcomas underwent radiosurgery (n = 42) or no radiosurgery (n = 45). Animals were sacrificed at 3, 6, 12, 24, 48, 72 h, and 1 or 2 weeks after treatment and in situ tumor apoptosis was assessed by specific staining. Tumor apoptosis was noted to be statistically higher in radiosurgery-treated animals relative to controls at the 6-, 24-, and 48-hour time points following radiosurgery. Radiosurgery induces apoptosis in the rat intracranial 9L gliosarcoma in a time-dependent fashion. The time course of this radiobiologic phenomenon begins at approximately 6 h following radiosurgery, continues up to 48 h, and begins to decline by 72 h.     

Testicular germ cell tumor seven years after a retroperitoneal germ cell tumor.

A 44-year-old male was diagnosed in August 1980 as having a retroperitoneal germ cell tumor (classic seminoma with anaplastic areas). After treatment with cisplatin-based chemotherapy, he reached complete clinical and pathological remission. Eighty-eight months later, in December 1987, he was diagnosed as having a testicular mixed germ cell tumor (embryonal carcinoma with anaplastic seminoma areas) after right orchiectomy. The potential mechanisms by which the latter tumor could have developed are discussed.     

Human plasma DNP level after severe brain injury

Objective: To determine the relationship between DNP level after human severe brain injury and hyponatremia as well as isorrhea. Methods: The peripheral venous plasma as control was collected from 8 volunteers. The peripheral venous plasma from 14 severe brain injury patients were collected in the 1,3,7 days after injury. Radioimmunoassay was used to detect the DNP concentration. Meanwhile, daily plasma and urine electrolytes, osmotic pressure as well as 24 h liquid intake and output volume were detected. Results: The normal adult human plasma DNP level was 62. 46 pg/ml±27. 56 pg/ml. In the experimental group, the plasma DNP levels were higher from day 1 to day 3 in 8 of the 14 patients than those in the control group (P1 =0.05, P3 =0.03). Negative fluid balance occurred in 8 patients and hyponatremia in 7 patients. The increase of plasma DNP level was significantly correlated with the development of a negative fluid balance (r = -0.69, P<0.01) and hyponatremia (X2=4.38, P<0.05). Conclusions: The increase of plasma DNP level is accompanied by the enhancement of natriuretic and diuretic responses in severe brain-injured patients, which is associated with the development of a negative fluid balance and hyponatremia after brain injury.     

Human rhabdosphincter cell culture: a model for videomicroscopy of cell contractions

BACKGROUND: Physiology of the human rhabdosphincter and its innervation are still a subject to controversy. A better understanding of rhabdosphincter function and anatomy might help to solve important urological problems like urinary incontinence. It was the aim of the present study to develop a human sphincter cell culture model for investigation of contraction mechanisms in vitro. METHODS AND RESULTS Cells were isolated from human rhabdosphincter tissue obtained from prostatectomy and cystoprostatectomy specimens. Cultured cells expressed typical features of striated muscle cells. By means of videomicroscopy with a time lapse videosystem cell contractions could be documented. Under control conditions without any contractile stimulant 8% of the cells were seen to contract. Cholinergic stimulation with 10 mM of acetylcholine induced a significant increase in contraction rate to 49%. CONCLUSIONS: These results demonstrate that cholinergic stimulation triggers contraction of cultured human rhabdosphincter cells. This model might help to understand external urethral sphincter physiology and to establish new therapies for the treatment of sphincter dysfunctions. Prostate 47:189-193, 2001.     

Differences in microvasculature between ethylnitrosourea-induced brain tumor and transplanted 9L cell brain tumor in the rat

The microvasculature of ethylnitrosourea (ENU)-induced brain tumors and transplanted 9L cell brain tumors were studied in ultrathin sections and by the freeze fracture replica method. The vessels in ENU-induced tumors were similar to human glioma vessels in that they had endothelial tight junctions and increased pinocytotic vesicles. In the 9L cell tumors, the vessels lacked endothelial tight junctions and had fenestrated endothelium. Macroscopically, Evans blue dye penetrated the 9L cell tumors but not the ENU-induced tumors. Judging from the ultrastructure of the microvessels, the ENU-induced tumor appears more suitable as a human glioma model.     

Treatment of metastatic brain tumor from renal cell carcinoma

Twenty-eight patients with metastatic brain tumor from renal cell carcinoma were treated at the National Cancer Hospital, Tokyo, between 1962 and March 1989. In 13 patients, the median time interval between the initial diagnosis and pulmonary metastasis was 18 months, and the interval between pulmonary metastasis and brain metastasis was 13 months. In 10 patients, whose initial diagnosis was pulmonary metastasis, the median interval between pulmonary metastasis and brain metastasis was also 13 months. There were 2 patients who presented brain metastasis initially. The median survival time from the diagnosis of brain metastasis was 17 months for the patients whose brain tumors were surgically resected, but only 4 months for the patients who didn't receive surgery. The median survival time of the patients who received postoperative radiation was 20 months, while it was 10.5 months for the patients who received radiation therapy alone. Repeated serial CT scans of 7 patients with measurable brain metastases revealed partial response (PR) to radiotherapy in 2 patients (28.6%), no change (NC) in 4 patients (57.2%), and progressive disease (PD) in one patient (14.3%). BrdU labeling indices of resected brain metastases were about 2%, and the doubling time calculated on repeated serial CT scans was about 20 days. As these lesions are rather resistant to radiotherapy and grow relatively slowly they should be resected as much as possible.