Sclerosing therapy of internal hemorrhoids with a novel sclerosing agent. Comparison with ligation and excision.

BACKGROUND AND AIMS: Patients with prolapsing internal hemorrhoids were treated with a novel sclerosing agent (OC-108), and the results were compared with surgery of ligation and excision. PATIENTS AND METHODS: This study included 20 years or older patients with prolapsing internal hemorrhoids who visited ten medical institutions in Japan from October 2000 to October 2002. Investigation on surgery was also performed. RESULTS: Comparing OC-108 and surgery in patients with third- and fourth-degree internal hemorrhoids according to the Goligher's classification, for which surgery has been generally indicated, at 28 days after treatment, the disappearance rate of prolapse was similar between OC-108 and surgery, 94% (75/80 patients) and 99% (84/85 patients), respectively. The 1-year recurrence rate was 16% (12/73 patients) in the OC-108 group, and this value was satisfactory because of its less invasive nature while it was more or less higher compared with 2% (2/81 patients) in the surgery group. The incidences of pain and bleeding were lower in the OC-108 group. CONCLUSIONS: OC-108 is a useful alternative treatment for hemorrhoids.

Interaction of soybean agglutinin with leukemic T-cells and its use for their in vitro separation from normal lymphocytes by lectin-affinity chromatography.

A procedure for separation of leukemic T-cells from normal lymphocytes, using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently coupled with soybean agglutinin (SBA), then served as an affinity probe for fractionation of mixture of normal lymphocytes and leukemic cells. Leukemic cell lines, derived from acute lymphoblastic leukemia (Jurkat, MOLT-4, RPMI-8402), were tested. The elution of normal lymphocytes was carried out by PBS(-). The leukemic T-cells, interacting with SBA, were removed by N-acetyl-D-galactosamine or low-concentration acetic acid. The type and viability of the separated cell fractions were analyzed by flow cytometry and fluorescent microscopy, using adequate fluorescent antibodies. The interaction of leukemic T-cells with free SBA, as well as with SBA-conjugated Sepharose beads, was examined fluorimetrically and visualized by fluorescent microscopy, using FITC-SBA as a marker. The rate of cell elution on SBA-affinity column decreased in order: normal > leukemic T-cells. Both normal lymphocytes and leukemic T-cells were removed in a mixture from SBA-free Sepharose 6MB by PBS(-) and were not fractionated discretely. The leukemic T-cells specifically interacted with SBA as well as with SBA-affinity adsorbent. In contrast, the normal lymphocytes did not interact with free SBA as well as with SBA-conjugated Sepharose beads in the concentrations applied. The method potentially combines a discrete cell fractionation with manifestation of a specific target cytotoxicity of SBA against leukemic T-cells, without any influence on normal lymphocytes.

Purification of normal lymphocytes from leukemic T-cells by lectin-affinity adsorbents - correlation with lectin-cell binding.

Utilization of leukemic T-cells from normal ones, using lectin-affinity adsorbents, is described. CNBr-activated Sepharose 6MB was covalently coupled to Soybean (SBA) or Dolichos Biflorus Agglutinins (DBA), then serves as an affinity probe for separation of leukemic T-cells from normal lymphocytes. The normal lymphocytes were removed almost completely by phosphate buffered saline (Ca(2+) and Mg(2+) free) (PBS(-)) from lectin-affinity column. More than 80% of the leukemic T-cells were retained on the lectin-affinity adsorbent, whereas another 10-15% were easily removed by PBS(-). There was a very good linear correlation between percent of cells, retained on the lectin-affinity adsorbent and percent of cells, interacting with the respective free lectin (r=0.97 for SBA, and r=0.93 for DBA). The viability of normal lymphocytes was not influenced after passing through the columns. In the case of leukemic T-cells - about 90% of the easily removed cells were dead, and another 10% were viable cells, non-interacting with DBA or SBA.

Fractionation of normal and leukemic T-cells by lectin-affinity column chromatography.

A method for rapid fractionation of normal and leukemic T-cells (Jurkat, RPMI-8402, MOLT-4), using lectin-affinity column chromatography, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently conjugated with Dolichos biflorus agglutinin (DBA) over 24 h. The normal cells were eluted by phosphate buffered saline (Ca(2+) and Mg(2+) free), while the leukemic T-cells, interacting with DBA, were removed by N-acetyl-D-galactosamine or by low-concentrated acetic acid as a mobile phase. The cell fractions were detected spectrophotometrically at 600 nm. The rate of cell elution decreased in the order: normal>leukemic T-cells. The viability and the type of separated T-cell fractions were characterized by flow cytometry, using adequate fluorescent antibodies. The interactions between leukemic T-cells and DBA-saturated Sepharose beads were examined by fluorescent microscopy, using fluorescent isothiocyanate-DBA as a fluorescent marker.

Common cell-surface antigens functioning in self-recognition reactions by both somatic cells and gametes in the solitary ascidian Halocynthia roretzi.

The "contact reaction" is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self-sterility in this species. To identify the receptors and target ligands involved both in self-recognition by somatic cells and self-discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N-linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.

Targeting and anti-tumor efficacy of liposomal 5'-O-dipalmitoylphosphatidyl 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine in mice lung bearing B16BL6 melanoma.

2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) is a potent anti-cancer agent, and we previously observed that liposomal formulation of 5'-O-dipalmitoylphosphatidyl derivative of CNDAC (DPP-CNDAC) is desirable for targeting. For targeting to pulmonary cancer, we investigated the in vivo behavior of liposomes containing DPP-CNDAC by a non-invasive method using positron emission tomography. Liposomes composed of DPP-CNDAC and cholesterol (DPP-CNDAC/CH liposomes) were markedly accumulated in mice lung bearing B16BL6 melanoma. In metastatic pulmonary cancer model, DPP-CNDAC/CH liposomes significantly reduced the lung colonization in a dose-dependent manner. The activity was significantly superior to conventional liposomal formulation or soluble CNDAC. These results suggest that DPP-CNDAC/CH liposomes are useful for metastatic pulmonary cancer.

Absence of linkage between radiosensitivity and the predisposing atp7b gene mutation for heritable hepatitis in the LEC rat.

The LEC rat is known to be a mutant strain that spontaneously develops heritable hepatitis due to copper accumulation, caused by mutation of the copper-transporting ATPase gene (Atp7b). Immunodeficiency and radiosensitivity have also been observed. Hayashi et al. extensively examined the radiosensitivity of the LEC rat and concluded that its hypersensitivity is controlled by a single autosomal gene. Furthermore, they suggested the possibility that it correlates to copper accumulation due to the Atp7b gene mutation, because ionizing radiation-induced hydroxyl radicals might act in concert with copper-induced hydroxyl radicals. In the present experiment, we analyzed linkage between radiosensitivity and the mutation responsible for hepatitis in F(1) animals of a cross with the F344 rat. Our results clearly demonstrated an absence of any significant association. In addition, partial dominance for radiosensitivity was observed, and radiosensitive (F(1) x LEC) backcross rats were twice as numerous as their radioresistant counterparts, suggesting the possibility of control by two or more recessive genes.

Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines.

The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin-a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin-a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application.

Interaction of the hemolytic lectin, CEL-III, with cultured human leukemic cell lines.

We studied interaction of CEL-III with cultured human leukemic cell lines and lymphocytes from normal adults by evaluating the extent of cytotoxicity and cytoagglutination. Among acute T lymphoblastic leukemia (T-ALL) cell lines, CEL-III displayed increased toxicity against different acute lymphoblastic leukemia (ALL) cell lines as a function of increasing differentiation stage. In the case of acute B lymphoblastic leukemia (B-ALL) cell lines, CEL-III showed strong cytotoxicity against relatively immature cell lines. We found that CEL-III was more toxic for ALL cell lines than leukocytes obtained from peripheral blood of healthy adults. Strong influence of the additional amount of calcium ion on the extent of cytotoxicity was observed. In addition, we describe a new way to evaluate the extent of cytoagglutination in "% of agglutinated cells". These findings make CEL-III a promising candidate in research for lectins which bind to and destroy only the targeted leukemic cells.

Cytotoxicity induced by Erythrina variegata serine proteinase inhibitors in tumor hematopoietic stem cell lines.

Based on the soluble MTT tetrazolium/formazan assay, we evaluated the cytotoxicity of Erythrina variegata proteinase inhibitors in some tumor hematopoietic stem cell lines. Among the proteinase inhibitors, EBI, which belongs to the Bowman-Birk family of inhibitors, was cytotoxic in relatively differentiated cells such as Molt4 and Jurkat derived from acute T lymphoblastic leukemia (T-ALL) cells specifically, but ETIa and ECI, which are classified into Kunitz family inhibitors, did not. It was suggested that the differences in the cytotoxicity might be due to the molecular size of the inhibitors. The succinylation of lysine residue(s) of EBI led to about 50% loss of the trypsin inhibitory activity as compared with the authentic EBI. When Molt4 cells were incubated with this derivative, no significant cytotoxicity was observed. This suggests that the proteinase inhibitory activity might be involved in the cytotoxicity in human tumor cell lines.

Spectroscopic analysis of the cytoagglutinating activity of abrin-b isolated from Abrus precatorius seeds against leukemic cells.

The cytoagglutinating activity of abrin-b, a toxic lectin isolated from Abrus precatorius seeds, against cultured cell strains derived from acute lymphoblast leukemia (ALL) was investigated by visible (VIS) spectroscopy. Upon addition of abrin-b, the turbidity at 600 nm of cell suspension decreased and this change could be recorded as the cytoagglutination curve. From this curve, the cytoagglutination velocity (CV) and cytoagglutination intensity (CI) of each cell strain was measured. Each cell strain showed the respective CV and CI values and the cell strains derived from the T cell line were strongly agglutinated by abrin-b compared with those derived from the B cell line. Further, it has become apparent that the cytoagglutinating activity increased with an increase in the order of the differentiation of cell strains.

Chemoprevention by dietary dehydroepiandrosterone against promotion/progression phase of radiation-induced mammary tumorigenesis in rats.

When pregnant rats received whole body irradiations with 260 cGy gamma-ray at day 20 of pregnancy, and were then implanted with a diethylstilbestrol (DES) pellet for an experimental period of 1 year under feeding of a control diet, a high incidence (96.2%) of mammary tumors was observed. Administration of dietary 0.6% dehydroepiandrosterone (DHEA) together with DES implantation significantly decreased the incidence (35.0%) of mammary tumors. The first appearance of palpable tumors in the DHEA-fed group was 4.5 months later than that in the control group. For clarification of the mechanism of the chemopreventive action, we measured hormone levels in the serum of DHEA-fed rats. In the DHEA diet rats, the concentration of estradiol-17 beta exceeded, by approximately 6-fold, that in the control rats, while the levels of progesterone and prolactin were decreased by 30 and 45%, respectively. Interestingly, DHEA feeding prevented DES-induced hypertrophy of pituitary glands and DES-induced high level of prolactin in pituitary glands detected by immunohistochemical studies, but stimulated the development of mammary glands more than that in control rats treated with DES alone. These findings suggest that DHEA has a potent preventive activity against the promotion/progression phase of radiation-induced mammary tumorigenesis. The mechanism of chemoprevention by change of endocrinological environment is discussed.

Relationship between stages of mammary development and sensitivity to gamma-ray irradiation in mammary tumorigenesis in rats.

Mature Wistar-MS rats were ovariectomized and treated with estradiol benzoate and/or progesterone. Control animals were treated with olive oil. The rats were then exposed to gamma-rays and implanted with a pellet of diethylstilbestrol. The incidence of mammary tumors in rats treated with estradiol benzoate or with progesterone was significantly higher than in rats in the non-treated control group, whereas, in rats treated with both estradiol benzoate and progesterone, the incidence was not significantly different from that in the controls. Histological examination of the mammary tumors showed 2 types of neoplasm: adenocarcinoma and fibroadenoma. Interestingly, over half of all the tumors in the rats treated with estradiol benzoate were adenocarcinomas, while fibroadenomas were mainly induced in the rats treated with progesterone or with both estradiol benzoate and progesterone. The expression of estrogen and progesterone receptors in the tumor tissues showed some differences according to whether the groups were treated with estradiol benzoate or with progesterone. Morphologically, mammary glands at irradiation showed well-developed lobuloalveoli in both the estradiol-benzoate-treated rats and in those rats treated with both estradiol benzoate and progesterone. This was consistent with the higher incorporation of [3H]thymidine into the DNA in the mammary glands of rats in both of these groups. Our findings suggest that a more advanced developmental stage of the mammary glands, dependent upon ovarian hormones, is related to a higher incidence of mammary tumors induced by irradiation.

Susceptibility of lactating rat mammary glands to gamma-ray-irradiation-induced tumorigenesis.

Lactating rats of the Wistar-MS strain were irradiated with 260 cGy of gamma rays 21 days after parturition (day 21). Diethylstilbestrol (DES) pellets were implanted one month after termination of nursing and were allowed to remain for one year. A significantly higher incidence (96.4%) of mammary tumors was observed in these rats irradiated during late lactation than in virgin irradiated animals (30.4%). A control group of lactating animals irradiated during late lactation but not treated with DES was also observed for one year; the final incidence of mammary tumors in this group was 35.3%. The latency period was shortest in the DES-treated group irradiated during late lactation. Histological examination showed that the mammary glands of lactating rats were highly developed, with alveoli filled with milk. Five days after weaning, there was degeneration of alveolar tissue, concomitant with a marked decrease in the concentration of estrogen and prolactin receptors. A considerable amount of epithelial tissue remained in the mammary glands during the process of atrophy. When the rats were irradiated 5 days after weaning, and then were treated with DES for one year, the incidence of mammary tumors was 73.3%, significantly higher than that in virgin irradiated rats. However, this incidence was not significantly different from that in animals irradiated during late lactation. These results suggested that the induction of mammary tumors by gamma irradiation before or after weaning was more dependent upon the stage of differentiation in mammary glands than upon the proliferative activity of epithelial cells, and that DES is essential as a promoter for radiation-induced mammary tumorigenesis.

Promotive effects of diethylstilbestrol, its metabolite (Z,Z-dienestrol) and a stereoisomer of the metabolite (E,E-dienestrol) in tumorigenesis of rat mammary glands pregnancy-dependently initiated with radiation.

Wistar-MS rats received whole body irradiation with 260 cGy gamma-rays at day 20 of pregnancy and then were treated with diethylstilbestrol (DES), E,E-dienestrol (E,E-DIES) or Z,Z-dienestrol (Z,Z-DIES) for 1 year. DES administration caused the highest incidence of mammary tumors with a concomitant reduction of gain in body weight. When E,E-DIES or Z,Z-DIES in pellet form was implanted, the incidence of tumors was significantly lower than that observed in rats treated with DES. To clarify the increased susceptibility to mammary tumorigenesis after DES administration we measured hormone levels in the serum of rats implanted with pellets containing derivatives of the synthetic estrogens. The serum prolactin concentration was significantly increased by DES administration. When E,E-DIES or Z,Z-DIES pellets were implanted the prolactin level was markedly reduced to 4.5% and 0.7% of that observed in DES-treated rats, respectively. In addition, the serum concentrations of estradiol-17 beta and progesterone in rats with Z,Z-DIES pellets were higher than those of rats with DES or E,E-DIES pellets. A large number of DES-induced mammary tumors were positive for both estrogen and progesterone receptors, but no tumors negative for both receptors were obtained. The findings suggest that DES acts directly on radiation-initiated mammary cells via binding with estrogen receptors and/or stimulates the secretion of prolactin from the pituitary glands.

Follow-up study of anti-hepatitis C virus antibodies in blood donors implicated in post-transfusion non-A, non-B hepatitis.

We retrospectively examined the antibodies to p22, a hepatitis C virus (HCV) nucleocapsid protein, and to c100-3, a HCV nonstructural protein, in donors whose blood was transfused to patients who later developed post-transfusion non-A, non-B hepatitis. Of 13 such blood donors, three seroconverted and three seroreverted with the anti-c100-3 test. In contrast, 12 of the 13 blood donors showed the same results at transfusion and follow-up, and one donor showed seroconversion with the anti-p22 assay. The follow-up study shows that the anti-p22 antibody test provides consistent results and is far more suitable for screening blood than the anti-c100-3 test.

Subcutaneous wire traction technique without CO2 insufflation for laparoscopic cholecystectomy.

One hundred laparoscopic cholecystectomies were performed since April 1991. Eleven patients were treated with a new technique without CO2 insufflation, using a traction device to elevate the right upper quadrant wall. Two Kirschner wires were introduced subcutaneously to permit the abdominal wall to be lifted for satisfactory laparoscopic view, as the gas insufflation technique yields. Preoperative evacuation of the intestines and intraoperative muscle relaxation are necessary for easy and successful cholecystectomy. Three cases were converted to laparotomy because of remarkably distended intestine due to incorrect endotracheal intubations. No complications related to subcutaneous wire traction technique were noted in this series. Subcutaneous wire traction technique provides a simpler, and possibly safer alternative to the gas insufflation technique.

Influence of the estrous cycle at gamma-ray exposure on radiation-induced mammary tumorigenesis in virgin rats.

Wistar rats received whole body irradiation with 260 cGy gamma-rays at 10 a.m. of individual phases of their estrous cycle and then had diethylstilbestrol pellets implanted for 1 year. When the radiation was given during di-estrus II, the highest incidence (73.3%) of mammary tumorigenesis was observed, and mean latency until the first tumor appearance was 8.5 +/- 0.7 months. Also, rats irradiated on estrus had significantly lower incidence (35.3%) of mammary tumors than those irradiated on pro-estrus and di-estrus II, but there was no significant difference in latency period. Iball's indices of total mammary tumors, fibroadenoma plus adenocarcinoma, were 14.3 +/- 0.9, 28.8 +/- 2.1, 23.3 +/- 0.8 and 12.2 +/- 0.2 in rats irradiated during di-estrus I, di-estrus II, pro-estrus and estrus respectively. The percentage of adenocarcinomas was comparatively uniform (25.0 to 34.8%) throughout the various phases of the estrous cycles. Also, Iball's indices calculated from adenocarcinoma indicated no significant differences between all groups. From our results, the highest incidence of mammary tumors arose in rats after irradiation at di-estrus II with minimum level of prolactin in serum. We discuss different mechanisms of radiation-induced and chemical-carcinogen-induced tumorigenesis of mammary glands.

Improved serodiagnosis of non-A, non-B hepatitis by an assay detecting antibody to hepatitis C virus core antigen.

We examined sequential serum samples from 12 patients with well-characterized posttransfusion non-A, non-B hepatitis who had an acute, resolving self-limited type of clinical course for the presence of antibody to the hepatitis C virus nucleocapsid (core) protein (p22) expressed by a recombinant baculovirus. These sera were simultaneously examined for antibody to the hepatitis C virus nonstructural protein (C100-3) that is presently used for blood screening worldwide. In three patients, both anti-p22 and anti-C100-3 antibodies were detected, but anti-p22 was detected much earlier. In four patients, only anti-p22 was detected. Two other patients were considered to be hepatitis C virus carriers who had been already infected with hepatitis C virus. In one patient, only anti-C100-3 was detected, and it was transient. In two patients, neither antibody was detected. Anti-p22 was detected in at least one of eight samples of transfused blood. Of the nine samples of donated blood that were positive for anti-p22, only four were positive for anti-C100-3. This new assay detecting the antibody to the p22 protein is thus useful for the serodiagnosis of non-A, non-B hepatitis in the acute phase and for blood screening.