Biomedical subjects

H Okada

Publications and source records attributed to H Okada.

At least 433 records · Page 24Linked to original sources

PubMed1996

Inhibitory effect of double transfection to xenoendothelial cells using both decay accelerating factor and homologous restriction factor 20 genes on complement dependent cytolysis.

Hyperacute rejection in discordant xenotransplantation occurs due to the complement activation via classical and/or alternative pathway. Regulator of complement activation (RCA) molecules inhibit species-specific complement dependent cytolysis. To confirm the effect of gene engineering using double RCA molecules on xenogeneic cells, the inhibitory effect on complement dependent cytolysis was compared between bovine aortic endothelial cells (BAEC) doubly transfected with both decay accelerating factor (DAF) and homologous restriction factor (HRF) 20 cDNA and BAEC singly transfected with DAF alone using retroviral vector. The positive percent expression of DAF and HRF20 antigen on double transfectant (BAEC/D+H) was 97.0% and 95.0%, respectively, whereas that of DAF antigen on single transfectant (BAEC/D) was 97.0%. After incubated with 25% human serum and anti-BAEC antibody, the viability of double transfectant (BAEC/ D+H) was significantly preserved, compared with that of single transfectant (BAEC/D). These findings demonstrated that xenoendothelial cells doubly transfected with both DAF and HRF20 cDNA could be protected from complement dependent cytolysis more effectively than those singly transfected with DAF alone in the presence of antixenoendothelial antibody.

Animals↗

PubMed1996

Insulin receptor mRNA in the substantia nigra in Parkinson's disease.

Neurotrophic effects resulting from the insulin/insulin receptor system have been recognized as important in determining the etiological basis of neurodegenerative disorders. In Parkinson's disease, selective neuronal loss in the substantia nigra is accompanied by decreased immunoreactivity of the insulin receptor as determined using immunohistochemical studies. We performed semiquantitative mRNA analysis by reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for human insulin receptor exon 22, which encodes a region of the beta subunit of the receptor serving as a tyrosine kinase domain. The relative levels of mRNA in the substantia nigra from Parkinson's brain tissues showed a marked depression compared with those of normal controls. Further investigations are needed to decide whether this is a primary, disease-specific alteration of gene expression or merely a secondary process.

Aged↗

PubMed1996

Local inflammation caused by a monoclonal antibody that blocks the function of the rat membrane inhibitor of C3 convertase.

The participation of membrane inhibitors in C-mediated inflammation in vivo was analyzed in rats with the use of mAbs against membrane inhibitors of C. 512Ag inhibits C3 convertase of homologous rat C and rat CD59 prevents the formation of homologous membrane attack complexes. The histologic distributions of 512Ag and CD59 in rat skin were essentially the same. However, although intracutaneous administration of mAb against 512Ag (anti-512Ag) in the form of F(ab')2 induced a remarkable inflammatory response visualized by exudation of Evans blue that had been previously injected i.v., the mAb against CD59 caused no inflammatory exudate. Furthermore, anti-512Ag F(ab')2 caused deposition of C3 followed by intense polymorphonuclear leukocyte infiltration and hemorrhage at the injection sites within 24 h. This reaction was completely abrogated by depletion of C. Therefore, spontaneous C activation on self cell membranes requires amplification at the C3 convertase step, which is restricted by membrane inhibitors such as 512Ag, thus preventing inflammation. A type II allergic response might preferentially occur at sites that exhibit an impairment of membrane inhibitor(s) of C3 convertase. However, although depletion of leukocytes by preadministration of cyclophosphamide did not reduce C3 deposition by anti-512Ag. Evans blue exudation was appreciably reduced, indicating that infiltrated leukocytes play a role in enhancing and prolonging inflammation initiated by local C activation.

Animals↗

PubMed1996

Cytolysis of malignant glioma cells by lymphokine-activated killer cells combined with anti-CD3/antiglioma bifunctional antibody and tumor necrosis factor-alpha.

With the aim of developing an effective immunotherapy for malignant glioma, glioma cells were incubated with tumor necrosis factor-alpha (TNF-alpha) to increase their susceptibility to lysis by lymphokine-activated killer (LAK) cells. Treatment with exogenous TNF-alpha induced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells. In addition, the cytolytic activity of LAK cells toward exogenous TNF-alpha treated glioma cells was significantly greater than LAK cell activity toward untreated glioma cells. This increase in cytolytic activity was blocked by anti-ICAM-1 monoclonal antibodies (MAb). Furthermore, co-treatment with a bifunctional antibody (BFA) composed of anti-CD3 (UCHT1) and antiglioma (G-22) antibodies synergistically increased the cytolytic activity of LAK cells towards TNF-alpha-treated glioma cells. These results indicate that a combination of exogenous TNF-alpha and anti-CD3/antiglioma BFA may provide an effective modified adoptive immunotherapy for patients with malignant glioma.

Antibodies, Bispecific↗

PubMed1996

Establishment of a human DAF/HRF20 double transgenic mouse line is not sufficient to suppress hyperacute rejection.

To solve the chronic donor organ shortage, the pig is considered to be a possible donor candidate for human transplantation. However, hyperacute rejection occurs due to the activation of the complement cascade. Therefore, the introduction of human complement inhibitors into animal cells has been proposed as a means to prevent such exologous complement activation. To investigate the extent to which complement inhibitors are resistant to human sera in discordant animals, we established transgenic mice lines which expressed either human decay-accelerating factor (DAF) and/or homologous restriction factor 20 (HRF20) using microinjection methods. Human sera were injected into (a) 10 control mice, (b) 10 DAF-transgenic mice, (c) 10 HRF20-transgenic mice, and (d) 10 DAF and HRF20-transgenic mice. The results showed that all the mice in groups a, b, and c died immediately after injection. Three of the mice in group d died, while seven survived but showed hyperpnea and low activity. The pathological findings of groups a, b, and c included severe coagulation; however, the survivors of group d showed less severe symptoms. The above findings thus suggest that both DAF and HRF20 tend to prevent complement activation to some extent; however, its effectiveness is not considered to be sufficient for clinical use in transplantation.

Animals↗

PubMed1996

The induction of cytotoxicity by a bispecific antibody against CEA positive cell line, in vitro.

A mouse anti-human carcinoembryonic antigen (CEA) x anti-human CD3 bispecific antibody, AB5C10*UCHT1, was developed. This antibody-heteroconjugate was chemically prepared by cross-linking the AB5C10 monoclonal antibody reactive with human CEA with the monoclonal antibody, UCHT1, which binds to CD3 on human T-lymphocytes. The AB5C10*UCHT1 recognized both CEA expressed on the KATOIII cell line and CD3 expressed on T-lymphocytes, as determined using flowcytometry. Next, AB5C10*UCHT1-mediated cytolysis was analyzed by 51Cr-release assay. When 51Cr-labeled target KATOIII cells were incubated for 6 h with effector cells that had been pretreated with AB5C10*UCHT1 for 60 min at 4 degrees C, the percentage specific lysis was significantly increased compared to that of untreated effector cells. Using peripheral blood mononuclear cells (PBMC) and lymphokine-activated killer (LAK) cells pretreated with AB5C10*UCHT1 for effector cells, the percentage specific lysis was determined to be 16.3% and 57.4% at effector: target (E:T) ratios of 100:1 and 12.5:1, respectively. On the other hand, the percentage specific lysis of untreated PBMC and LAK cells determined to be 3.0% and 35.8% at E:T ratios of 100:1 and 12.5:1, respectively. The minimum effective dose of AB5C10*UCHT1 required for antibody-mediated cytotoxicity was 0.1 mu g/ml. The results of this study suggest that AB5C10*UCHT1 could be useful for augmenting the cytotoxicity of CD3-positive T-cells against CEA-positive target cells in vitro.

Animals↗

PubMed1996

In vitro model of parathyroid hormone-related protein secretion from mammary cells isolated from lactating cows.

Parathyroid hormone-related protein (PTHrP) is produced by the lactating mammary gland and is present in milk in a biologically active form. The goal of this investigation was to determine if cells cultured from the lactating mammary glands of cows would secrete PTHrP in vitro. Mammary acini were isolated from lactating cows at 1-6 wk after calving, and fresh or cryopreserved mammary acini were cultured for 14 d on Type I collagen. Cultures on thick layers of collagen (2.5 mm) were detached and allowed to contract on Day 6. PTHrP production was measured by N-terminal radioimmunoassay and bioassay (increased cAMP levels in ROS 17/2.8 osteoblast-like cells). The mammary cells reached confluence at Day 6. PTHrP production was low at Day 2 (< 0.5 ng/ml) but increased to peak production (2-4 ng/ml) at approximately Day 6 and remained constant until Day 14. Immunoreactive and bioactive PTHrP levels in the culture medium correlated well. The cultures produced lactoferrin (2,000-2,300 ng/ml and alpha s1-casein (14-19 ng/ml). Prolactin stimulated PTHrP production approximately 50% on Days 6-14. PTHrP production was increased approximately 100% by treatment with epidermal growth factor (10 ng/ml) for 2 d. Morphologic evaluation of cultures on thick, contracted collagen at Day 14 revealed an inner layer of mammary epithelial cells overlying myoepithelial cells and an outer layer of collagen containing stromal cells. Immunohistochemistry demonstrated positive staining for PTHrP and cytokeratin in both mammary epithelial and myoepithelial cells and alpha-smooth muscle actin in myoepithelial cells. These data demonstrated that cryopreserved mammary tissue from lactating cows could be cultured in vitro and secreted PTHrP in a regulated manner. This in vitro model will be useful to investigate the function and regulation of PTHrP in the lactating mammary gland.

Adenylyl Cyclases↗

PubMed1996

Development of a quantitative sampling method for periapical exudates from human root canals.

A quantitative method for collection of periapical exudates on endodontic paper points is described. In a pilot study, it was revealed that the relationship between fluid volume and wetted length of paper points had a highly significant curvilinear relationship (p < 0.0001), and a highly significant positive linear relationship was found to describe eluted and absorbed interleukin (IL)-1 beta activities from paper points. Periapical exudates from 29 root canals with apical periodontitis were collected using this method, and IL-1 beta activities in clinical specimens were measured. Periapical exudates (0.15 to 26.7 microliters) were recovered, and the range of IL-1 beta concentration was 0.1 to 179.5 ng/ml. These results showed that this sampling method was useful to analyze immunological changes in periapical lesions.

Dental Pulp Cavity↗

PubMed1996

Relationship between prostaglandin E2 concentrations in periapical exudates from root canals and clinical findings of periapical periodontitis.

In this study the relationship of prostaglandin E2 (PGE2) concentrations in periapical exudates with clinical findings of teeth with periapical periodontitis is discussed. Periapical exudate samples were obtained from root canals of 77 endodontically involved teeth during routine root canal treatment by the quantitative sampling method using paper points. PGE2 concentrations in periapical exudates (PE-PGE2) were determined by radioimmunoassay. Significantly higher levels of PGE2 were found in periapical exudates from teeth with radiolucent areas than from teeth without radiolucent areas (236.8 +/- 521.3 pg/microliter vs. 14.9 +/- 23.1 pg/microliter, respectively). The elevated PE-PGE2 levels were associated with the presence of clinical symptoms that reflected an acute inflammation in the periapical lesion. In contrast, a significantly negative association of decreased PE-PGE2 levels with increasing size of radiolucent areas was demonstrated. These results suggested that PGE2 was produced locally in periapical lesions and that the PGE2 concentration in periapical exudate could reflect the state of the disease activity in periapical periodontitis.

Chi-Square Distribution↗

PubMed1996

Laparoscopic-assisted creation of a vagina.

The applicability of laparoscopy for colpopiesis using pelvic peritoneum was examined in two women with the Mayer-Rokitansky-Kuster-Hauser syndrome. Using a modification of laparoscopic guidance proved advantageous for safe dissection of the vesicorectal space and accurate suturing of the pelvic peritoneum without laparotomy. A sufficient vaginal cavity was achieved in both patients. This modification of the original procedure, featuring introduction of a laparoscope, thus provides effective and safe conditions for creation of a new vagina.

Adult↗

PubMed1996

Magnetic resonance appearance of bone marrow in the mandible at different ages.

To differentiate pathologic states of bone marrow from normal age-related marrow conversion, magnetic resonance appearances of the mandible were analyzed cross-sectionally according to age in 73 normal volunteers. Low signal intensities of red marrow first changed to high signal intensities in the anterior region of the mandibular body. With increase in age, marrow conversion was observed in the premolar/molar region, angle, ramus, and condyle regions in that order. No red marrow was recognized in the body of the mandible of subjects more than 30 years old. After the age of 30, the majority of the subjects showed high signal intensities in the entire bone marrow of the mandible. However, one subject over 30 years of age showed some portions of red marrow in the ramus and condyle. This knowledge about age-related marrow conversion of the mandible on magnetic resonance imaging will be useful in the visual differentiation from abnormal bone marrow, such as anemias, inflammatory diseases, infiltrative neoplastic disorders, and metastatic diseases.

Adolescent↗

PubMed1996

Persistent suppression of the pituitary-gonadal system in female rats by three-month depot injectable microspheres of leuprorelin acetate.

The serum drug levels and pharmacological effects of leuprorelin acetate (leuprolide acetate) in female rats after subcutaneous (sc) injection of 3-month release injectable and biodegradable microspheres were investigated. After sc injection, the microspheres provided sustained serum drug levels for > 3 months, probably because of continuous release of the drug. As was found for the 1-month depot preparation, a transient initial high peak in serum leuprorelin levels resulting from an initial burst of drug release could not be avoided. Sustained suppression of serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), and estradiol for > 16 weeks was attained by a single injection of the microspheres. An injection of the microspheres suppressed the growth of the ovaries and uterus in a dose-dependent manner; a drug dose of 0.036 mg (corresponding to approximately 1 microgram/kg/day) was insufficient for suppression of uterine growth, and a drug dose of 3.6 mg (corresponding to approximately 100 micrograms/kg/day) provided the strongest growth suppression in both organs. Serum LH, FSH, and estradiol responses in a challenge test indicated that definite pharmacological effects were sustained for at least 16 weeks, as was demonstrated previously in male rats and dogs. Thus, this depot formulation may provide sustained therapeutic effects for 3 or 4 months in humans.

Animals↗

PubMed1996

Distribution of activated complement, C3b, and its degraded fragments, iC3b/C3dg, in the colonic mucosa of ulcerative colitis (UC).

The third component of complement (C3) is central to both the classical and alternative pathways in complement activation. In this study, involvement of C3 activation in the mucosal injury of UC was investigated. We examined the distribution of activated (C3b) and degraded fragments (iC3b/C3dg) of C3, terminal complement complex (TCC), and complement regulatory proteins in normal and diseased colonic mucosa including UC and other types of colitis using immunohistochemical techniques at the level of light and electron microscopy. While C3b and iC3b/C3dg staining was negligible in the normal mucosa, iC3b/C3dg and, to a lesser extent, C3b were deposited in UC mucosa along the epithelial basement membrane. The deposition was enhanced in relation to the severity of mucosal inflammation (C3b, P less than 0.05; iC3b/C3dg, P less than 0.01). Epithelial deposition of TCC was not observed in most UC mucosa. Immunoelectron microscopy showed that C3b and iC3b/C3dg were distributed mainly along the epithelial basement membrane and the underlying connective tissue in a granular, studded manner, and weakly present along the basolateral surface of epithelial cells. These C3 fragments were also deposited in inflammatory control mucosa such as ischaemic and infectious colitis. Our findings suggest that deposition of the C3 fragments occurs in inflamed colonic mucosa of diverse etiologies, including UC, but to define a role of the deposition in the development of mucosal injury in UC awaits direct study.

Adult↗

PubMed1996

Complement plays an essential role in shock following intestinal ischaemia in rats.

Intestinal ischaemia lasting more than 30 min in rats causes fatal systemic shock. Systemic shock was suppressed by preadministration of cobra venom factor (CVF), which reduced the serum complement to less than 5% of the normal level, indicating that complement is involved in the syndrome. After complement activation, anaphylatoxins such as C3a and C5a are generated, and their activity is restricted by carboxypeptidases which remove C-terminal arginine from such bioactive peptides. As expected, preadministration of a carboxypeptidase inhibitor enhanced the systemic shock induced by the intestinal ischaemia. However, when the complement level was suppressed by CVF treatment, no fatal systemic shock was induced by the intestinal ischaemia even with preadministration of the carboxypeptidase inhibitor. These results indicate that complement plays a crucial role in systemic shock induced by intestinal ischaemia, and that anaphylatoxins generated by the complement activation should be involved in induction of the shock syndrome.

3-Mercaptopropionic Acid↗

PubMed1996

Assessment of germ-cell kinetics in the testes of patients with varicocele using image analysis of immunostained proliferating cell nuclear antigen.

OBJECTIVE: To examine the germ-cell kinetics of varicocele testis using proliferating cell nuclear antigen (PCNA) immunostaining and image cytometry to evaluate the staining intensity, and to compare the proliferative index (PI) of varicocele and normal testes. PATIENTS AND METHODS: Fifty-six testicular biopsy specimens were taken from 28 patients with varicocele during high ligation. The specimens were fixed and embedded in paraffin, and sections immunostained using an anti-PCNA antibody. The PI was measured using an image analyser and expressed as the percentage of the total nuclear area stained positively with PCNA monoclonal antibody, assessed from > 20 seminiferous tubules. Sections stained with haematoxylin and eosin were used to determine Johnsen's score using conventional microscopy. Normal control testicular biopsies were obtained from subjects undergoing vasectomy. RESULTS: In patients with varicocele, both testes had a significantly lower PI than normal testes; the mean (SD) PI of the right and left testes was 11.3% (4.1) and 11.3% (4.8), respectively, and the PI of normal testes was 21.7% (3.1). There was no significant correlation between PI and sperm concentration or Johnsen's score. CONCLUSION: PI is useful for assessing germ-cell kinetics; in patients with varicocele both testes showed a deterioration in DNA synthesis, suggesting that reduced DNA synthesis is one of the causes of spermatogenic dysfunction in the varicocele testes.

Adult↗

PubMed1996

IgM natural antibody against an asialo-oligosaccharide, gangliotetraose (Gg4), sensitizes HIV-I infected cells for cytolysis by homologous complement.

Although cells are usually resistant to homologous complement, the IgM antibody against gangliotetraose (Gg4), an asialo-oligosaccharide of GM1, was found to cause cytolysis of HIV-1 infected cells by homologous complement. Due to its size, IgM might enable the initiation of the complement cascade away from membrane complement inhibitors such as decay accelerating factor and membrane cofactor protein. Furthermore, HRF20 (CD59), which restricts formation of membrane attack complexes (MAC) of complement on homologous cell membranes, was significantly decreased on HIV-infected cells and therefore formation of MAC on cell membranes would be facilitated. IgM antibodies reactive with HIV-infected cells could result in the elimination of infected cells via complement-mediated cytolysis in HIV-infected patients, since all tested sera from HIV-positive hemophilia patients who have survived for >12 years contained IgM antibody activity against HIV-infected MOLT4 cells in a preliminary experiment. Therefore, administration of IgM antibodies reactive with HIV-infected cells may be effective in the treatment of HIV carriers.

CD4-Positive T-Lymphocytes↗

PubMed1996

Molecular cloning of murine decay accelerating factor by immunoscreening.

Although the cDNA of human decay accelerating factor (DAF) which restricts complement activation on homologous cell membranes was cloned in 1987, all trials to detect the cDNA of mouse DAF by cross-hybridization were unsuccessful. However, by immunoscreening with a rabbit antiserum against purified mouse DAF, we successfully cloned the cDNA. It contains four typical short consensus repeats (SCR) similar to that in human and guinea pig DAF. The base sequence showed 63.7 and 63.8% identity to that of human and guinea pig DAF respectively. The deduced amino acid sequence identity to human and guinea pig DAF was 47.2 and 46.5% respectively. Mouse complement receptor related gene Y (Crry)/p65 function is comparable to DAF. SCR3 and SCR4 of mouse DAF showed 50% identity to SCR2 and SCR3 of Crry/p65 respectively. Identification of the mouse DAF gene should open a new approach for determining the actual in vivo role of DAF by analyzing autoimmune mice as well as generating DAF gene knockout mice using embryonic stem cells.

Amino Acid Sequence↗

PubMed1996

Calcium channel blockers versus ACE inhibitors as antihypertensives in polycystic kidney disease.

The effects of calcium channel blockers (CCBs) and angiotensin converting enzyme (ACE) inhibitors on blood pressure and the progression of renal dysfunction were compared in hypertensive patients with polycystic kidney disease (PKD). Twenty-six patients with PKD and hypertension who had been treated with other antihypertensive agents, such as diuretics, beta-blockers, or alpha-methyldopa, were followed up for two years, during which their blood pressure and renal function were monitored. Patients were divided into two groups classified according to the type of antihypertensive agents given. Group 1 (n = 14) received CCBs, while group 2 (n = 12) received ACE inhibitors. No significant differences were found in their blood pressure control and serum creatinine levels throughout the study. The creatinine clearances were decreased in both groups. However, the decreases in creatinine clearance were smaller (p < 0.05) in the group treated with CCBs. In addition, two patients in group 2 showed rapid increases in serum creatinine. Our data suggest that CCBs reduced blood pressure effectively and preserved renal function in PKD patients at least as well as ACE inhibitors.

Angiotensin-Converting Enzyme Inhibitors↗