Development of pro-apoptotic peptides as potential therapy for peritoneal endometriosis.

Endometriosis is a common gynaecological disease associated with pelvic pain and infertility. Current treatments include oral contraceptives combined with nonsteroidal anti-inflammatory drugs or surgery to remove lesions, all of which provide a temporary but not complete cure. Here we identify an endometriosis-targeting peptide that is internalized by cells, designated z13, using phage display. As most endometriosis occurs on organ surfaces facing the peritoneum, we subtracted a phage display library with female mouse peritoneum tissue and selected phage clones by binding to human endometrial epithelial cells. Proteomics analysis revealed the z13 receptor as the cyclic nucleotide-gated channel ß3, a sorting pathway protein. We then linked z13 with an apoptosis-inducing peptide and with an endosome-escaping peptide. When these peptides were co-administered into the peritoneum of baboons with endometriosis, cells in lesions selectively underwent apoptosis with no effect on neighbouring organs. Thus, this study presents a strategy that could be useful to treat peritoneal endometriosis in humans.

A genome-wide linkage analysis of orchard grass-sensitive childhood seasonal allergic rhinitis in Japanese families.

Seasonal allergic rhinitis (SAR) is an inflammatory disease of the nose and eyes that follows sensitization to air-born pollens. We conducted a genome-wide linkage screening of 48 Japanese families (188 members) with orchard grass (OG)-sensitive SAR children (67 affected sib-pairs) in a farming community in central Japan where OG was planted for apple farming and OG pollen is a major cause of SAR. We used the GENEHUNTER program to performed nonparametric multipoint linkage analysis for OG-sensitive SAR as a qualitative trait and for log total serum IgE levels and OG-RAST IgE levels as quantitative traits. Genotyping data of 400 microsatellite markers suggested linkage of SAR to chromosomes 1p36.2, 4q13.3, and 9q34.3 (P < 0.001), linkage of serum total IgE levels to 3p24.1, 5q33.1, 12p13.1, and 12q24.2 (P < 0.001), and linkage of OG-RAST IgE levels to 4p16.1, 11q14.3, and 16p12.3 (P < 0.001). Weak evidence for linkage of SAR to 5q33.1 was also observed (P = 0.01). All these regions, with the exception of 9q34.3, have been previously reported to be linked to asthma and/or atopy. These data suggest that, although loci linked to SAR are likely to be common to asthma, a strong contribution by specific gene(s) to OG-sensitive SAR is unlikely.

[Evaluation of dermatological symptoms of Yusho patients in the annual examination in 1999-2000].

We analyzed the severity grades of the skin symptoms of Yusho patients who visited the annual examinations in 1999 and 2000. The severity grades of the skin symptoms clearly improved. The patients graded as 0 increased and those graded as II.III-III or III.IV-IV decreased as compared to the data in 1993. The pattern of the skin severity scores did not change much, although the patients who showed 0.1 reached 68% in 2000. The blood PCB concentration of Yusho patients also clearly decreased, especially in the patients who showed "A" pattern. Furthermore, the skin changes of the aged patients should be analyzed in detail in the future.

Induction of CD54 and CD58 expression in cultured human endothelial cells by beta-interferon with or without hyperthermia in vitro.

Human endothelial cells were treated by beta-interferon with or without hyperthermia at 42 degrees C for 90 min in vitro to investigate whether these modalities were able to increase the expression of either CD54 or CD58 on the surface of the endothelial cells. The results were that the population of the endothelial cells expressing both CD54 and CD58 increased 4 days after the treatment with beta-interferon, which was independent of hyperthermia. In contrast, the primarily isolated peripheral lymphocytes from a patient with malignant melanoma (disease free state) or normal individuals responded to neither beta-interferon nor hyperthermia in terms of the expression of CD54 or CD58. These results indicate that beta-interferon may activate endothelial cells to lead to the successive activation of the other immune cells in vivo.

Systemic effects of hyperthermic isolated lower limb perfusion with carboplatin and interferon-beta.

The changes in systemic circulation during hyperthermic isolated lower limb perfusion with carboplatin and interferon-beta were investigated in 19 patients with malignant melanoma. The cardiac output (CO) increased significantly (p < 0.01) from 3.81 +/- 0.22 L/min before the procedure to 5.30 +/- 0.49 L/min 1 h after hyperthermic perfusion. The double product (mean arterial pressure x heart rate) also increased significantly (p < 0.01) from 5,145 +/- 372 mm Hg/min to 6,760 +/- 486 mm Hg/min. In some patients, it increased to more than twice the control value. These changes were accompanied by an increase in body temperature, presumably caused by the systemic leakage of both warmed blood and interferon-beta. Blood chemistry data demonstrated no significant changes in the liver or renal function. However, the serum CPK level increased markedly on the first postoperative day, and persisted for 1 week, thus suggesting that some muscle damage occurred during the procedure. There was no operative death or severe complications. From these data, we concluded that hyperthermic isolated limb perfusion with interferon-beta is a relatively safe therapeutic method for malignant melanoma of the extremities. However, care should be taken in patients with ischemic heart disease who may suffer a heart attack due to the rapid increase in cardiac work during the procedure.

Current techniques of hyperthermic isolated limb perfusion for malignant melanoma.

A retrospective study was conducted examining 25 patients with malignant melanoma who were treated by our new protocol for hyperthermic isolated limb perfusion. The characteristics of our techniques include: a lower priming volume of the extracorporeal circuit; a therapeutic temperature range of 40-41 degrees C with 60 min hyperthermic perfusion; a nominal perfusion flow rate of 500 ml/min in the lower limb and 200 ml/min in the upper limb; and combined carboplatin with interferon-beta as the adjuvant chemotherapy drug. In the lower extremity group, the arterial cannula size ranged from 8 to 14 F, while the venous cannula size ranged from 14 to 16 F. In the upper limb group, the arterial cannula size ranged from 6 to 8F and the venous cannula size ranged from 10 to 12F. No patient required any homologous blood transfusion postoperatively. No operative death or major complications occurred during the early postoperative period, confirming the safety of this treatment. Both optimal cannula size selection and maintaining perfusion temperature below 41 degrees C were judged to be important in elimination of vascular and deep tissue injury.

A novel, high endothelial venule-specific sulfotransferase expresses 6-sulfo sialyl Lewis(x), an L-selectin ligand displayed by CD34.

L-selectin mediates lymphocyte homing by facilitating lymphocyte adhesion to unique carbohydrate ligands, sulfated sialyl Lewis(x), which are expressed on high endothelial venules (HEV) in secondary lymphoid organs. The nature of the sulfotransferase(s) that contribute to sulfation of such L-selectin counterreceptors has been uncertain. We herein describe a novel L-selectin ligand sulfotransferase, termed LSST, that directs the synthesis of the 6-sulfo sialyl Lewis(x) on L-selectin counterreceptors CD34, GlyCAM-1, and MAdCAM-1. LSST is predominantly expressed in HEV and exhibits striking catalytic preference for core 2-branched mucin-type O-glycans as found in natural L-selectin counterreceptors. LSST enhances L-selectin-mediated adhesion under shear compared to nonsulfated controls. LSST therefore corresponds to an HEV-specific sulfotransferase that contributes to the biosynthesis of L-selectin ligands required for lymphocyte homing.

[Evaluation of dermatological symptoms of Yusho patients in the annual examination in 1997-1998].

We analyzed the severity grades of the skin symptoms of Yusho patients who visited the annual examinations in 1997 and 1998. The severity grades of the skin symptoms clearly improved. The patients graded as 0 I-II increased and those graded as II III-III decreased as compared to the data in 1993 and 1994. The skin severity scores did not change much, although the patients who showed 0 or 1 reached more than 60% in both 1997 and 1998. The blood PCB concentration of Yusho patients also clearly decreased, especially in the patients who showed "A" pattern.

Increase in the peripheral lymphocyte populations expressing CD54 (ICAM-1) after hyperthermic isolated limb perfusion in patients with malignant melanoma: an analysis of four cases.

The lymphocytes isolated from perfused or non-perfused circulations before, during, and after hyperthermic isolated limb perfusion (HILP) in the four patients with malignant melanoma were analysed for the expression of CD54 (ICAM-1), CD58 (LFA-3), CD4, CD8, HLA class I and class II in order to investigate the mechanism(s) of the activation of such immunocompetent cells as natural killer (NK)-cells or T-lymphocytes by HILP. It was thus found that the lymphocyte populations expressing CD54 increased significantly 1 day after HILP in the four patients examined. The lymphocyte populations expressing CD58 apparently increased. It was also found that the NK-cell and T-lymphocyte activities increased during or after HILP in the present four cases as observed previously in the other melanoma patients. These results indicate that our HILP system may augment the immunological activities through the mechanisms of the induction of CD54 or CD58 expression in the peripheral lymphocytes of the melanoma patients who receive HILP.

Experimental approaches for the treatment of murine B16 melanomas of various sizes. I: Local injection of ethanol with a combination of interleukin-2 or microwaval hyperthermia for B16 melanomas with a size of less than 7 mm in diameter.

The effects of local absolute ethanol injection combined with administration of interleukin-2 (IL-2) or microwaval hyperthermia in murine B16 melanomas with a size of approximately 7 mm in diameter were investigated. The groups of melanoma-burdened mice treated with both local ethanol injection and local or intra-abdominal administration of IL-2 showed clear suppression of any recurrence of melanoma once the melanomas had been destroyed by ethanol injection and a concomitant prolongation of the survival times. Also, local injection of ethanol in combination with local microwaval hyperthermia at 43 degrees C for 15 min twice a week caused complete cures in B16 melanomas with a size of less than 7 mm in diameter. The infiltrations of T lymphocytes and NK cells were augmented in the melanomas treated with ethanol injection and local injection of IL-2. However, the melanomas treated with ethanol injection and intra-abdominal injection of IL-2 hardly showed any infiltration of such immune cells, although the growth of melanomas was effectively suppressed. In the case of treatment with ethanol and hyperthermia, slight infiltration of NK cells was observed in the melanoma nests as well as in the interstitials. Thus, the direct injection of absolute ethanol in combination with IL-2 or microwaval hyperthermia is effective or even curative in the treatment of murine B16 melanomas with a size of less than 7 mm in diameter.

Experimental approaches for the treatment of murine B16 melanomas of various sizes. II: Injection of ethanol with combinations of beta-interferon and microwaval hyperthermia for B16 melanomas with a size of greater than 10 mm in diameter.

Although ethanol injection in combination with microwaval hyperthermia and systemic administration of interleukin-2 was found to be very effective in suppressing the growth of B16 melanoma nodules with a size of less than 7 mm in diameter in our previous experiments, the growth of B16 melanomas with a size of greater than 10 mm in diameter was very difficult to control with any of the therapeutic modalities examined. In subsequent experiments, we investigated whether ethanol injection (150 microliters/injection) in combination with local injection of beta-interferon (2 x 10(4) IU/injection) and local microwaval hyperthermia at 43 degrees C for 15 min was able to suppress the growth of melanoma nodules and to prolong survival times of melanoma-burdened mice. The results were that we successfully suppressed the growth of melanoma nodules of that size for the first time with combinations of the therapeutic modalities described above. Infiltration of immunocompetent cells such as T lymphocytes and NK cells was clearly observed in the melanoma tissues treated with the therapeutic combination. These experimental results should be applied to the treatment of human melanomas in advanced stages which have no indications for surgical operation.

New nortriterpenoid isolated from anti-rheumatoid arthritic plant, Tripterygium wilfordii, modulates tumor growth and neovascularization.

Preparations of Tripterygium wilfordii, "Thunder God vine", have been used in China to treat rheumatoid arthritis. Rheumatoid arthritis, as well as solid tumors, is closely associated with neovascularization. Antiarthritic drugs therefore may modulate tumor growth as well as neovascularization. We found that a compound purified from T. wilfordii, the nortriterpenoid, demethylzeylasteral (TZ-93), inhibited the proliferation of vascular endothelial cells approximately 30 times more effectively than it did for the proliferation of human tumor cells. In in vitro assays using bovine aortic endothelial cells, TZ-93 at non-toxic doses inhibited cell migration, expression of urokinase-type plasminogen activator (uPA) mRNA and uPA activity. Exogenous addition of uPA restored the inhibitory effect of TZ-93 on cell migration. In dorsal air-sac assays in BALB/c mice, the oral administration of 3 mg/kg/day TZ-93 for 5 days partially inhibited, and 30 mg/kg/day almost completely abrogated, the development of capillary networks induced by human hepatoblastoma cells. Similarly, 0.3 mg/kg/day TZ-93 partially inhibited, and 3 or 30 mg/kg/day almost completely blocked, the growth of mouse B16-F10 melanoma cells in a tumor implantation assay. The highest dose of TZ-93 significantly reduced the growth of well-vascularized tumors with volumes of more than 500 mm3. TZ-93 treatment of tumor-bearing mice significantly decreased the density of microvessels in the tumors. We conclude that TZ-93 may be useful in treating highly vascularized and metastatic tumors as well as other angiogenic diseases.

Kinetics of immunological parameters in patients with malignant melanoma treated with hyperthermic isolated limb perfusion.

Kinetics of immunological parameters such as natural killer (NK) cell activity and tritium-thymidine uptake rate of T lymphocytes by phytohemagglutinin stimulation were investigated using peripheral leukocyte fractions of melanoma patients treated with hyperthermic isolated limb perfusion (HILP). Also, serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) were quantified during and after HILP. It was found that NK cell activity was augmented during HILP, and T lymphocyte function was stimulated 24 h and 1 week after HILP with statistical significance. NK cell activities in the cells isolated from perfused and non-perfused circulations were equally augmented during HILP in two patients examined. Serum concentrations of sICAM-1 in the patients who received HILP also increased 24 h or even 1 week after HILP. The stimulation of these immune competent cells and upregulation of sICAM-1 by HILP were independent of the stages of melanoma patients at the time of HILP or the doses of agents which were used for the infusion during HILP. The origin of cells which shed sICAM-1 into the serum of the patients who received HILP remains to be further investigated.

[Levels of PCDDs, PCDFs and coplanar PCBs in sebum and blood of Yusho patients].

Polychlorinated dibenzo-p-dioxins (PCDDs) and it's related compounds such as polychlorinated dibenzofurans (PCDFs) can be detected in the body in Yusho patients, a condition caused by ingestion of contaminated rice oil with these compounds. These compounds are excreted out of the body in small amounts directly in the feces from the wall of the intestine. We found the PCDDs, PCDFs and coplanar polychlorinated biphenyls (Co-PCBs) in the sebum of Yusho patients and normal subjects, and there was significant correlation between their concentrations in sebum and blood in both patients and normal control subjects. The concentration of TEQ (toxic equivalent quantity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: TCDD) in sebum of Yusho patients (106 pg/g lipid) was half that in the blood (215 pg/g lipid), while that of normal subjects (29 pg/g lipid) was similar to that in the blood (34 pg/g lipid). On the other hand, in Yusho patients the concentrations of TEQ in the sebum and in blood were 2.3 and 6.4 fold higher than those in normal subjects. We concluded that dioxins and it's related compounds in Yusho patients and normal subjects were excreted not only in the feces but also in the sebum.

[Dermatological findings in the annual examination of the patients with Yusho in 1995-1996].

We reported the grades of severity of skin symptoms and the blood PCB patterns and concentrations in Yusho patients who had the annual examinations in 1995 and 1996. The skin severity grades and the skin severity scores showed the same tendency as recorded in the last 10 years. Yusho patients who were scored as more than 6.7 were around 8% of the total patients both in 1995 and 1996. The correlation between the blood PCB patterns and the severity grades of skin symptoms was clear. Thus, the mean skin severity scores in patients showing A pattern were higher than those in patients showing either B or C pattern. The same results were obtained when the correlation between the blood PCB patterns and the skin severity grades was analyzed. Patients whose ages were 50-70 years old were found to show the highest blood PCB concentrations and the skin severity scores.

Increase in shedding of intercellular adhesion molecule-1 in human malignant melanoma cell lines treated with hyperthermia in vitro.

Human malignant melanoma cell lines were found to increase shedding of soluble intercellular adhesion molecule-1 (sICAM-1) into the culture medium when the cells were treated with hyperthermia at 41-43 degrees C for 3-6 hr in vitro. The content of ICAM-1 in the cell lysate was also found to be increased after hyperthermia. The increased rate of ICAM-1 concentration in the cells was at maximum when they were incubated at 41 degrees C for 3 hr. Also, the melanoma cell lines heat-treated at 41 degrees C showed more intense immunofluorescence in the ICAM-1 expression on the cell surface. It remains to be investigated further whether the effects of hyperthermia on the ICAM-1 expression in melanoma cells is to augment membrane ICAM-1 expression, which in turn leads to shedding of soluble ICAM-1 or only to acceleration of shedding of sICAM-1 by unknown mechanisms.

Two cases of metastatic malignant melanoma of the lower limb treated with hyperthermic isolated limb perfusion and concomitant infusion of either carboplatin or beta-interferon.

Two cases of metastatic malignant melanoma of the lower limb who were treated successfully with hyperthermic isolated limb perfusion are reported. One patient was infused with cisdiammine (1.1-cyclobutanedicarboxylate) platinum (II) (carboplatin, Paraplatin, Bristol-Myers Squibb Company, New Jersey, USA), and the other was infused with human natural beta-interferon (Feron, Toray, Tokyo, Japan), via the external iliac artery. The first case showed a remarkable suppression of the growth of multiple metastatic melanoma nodules associated with numerous melanophage infiltrations, as shown histopathologically after the operation. The patient's serum level of 5-S-cysteinyl dopa decreased for the two months following the treatment. In the second case, new formation of metastatic melanoma nodules was completely suppressed for up to 12 months following the operation. Analysis of immunological parameters showed that the number of peripheral CD8+ lymphocytes gradually and constantly increased after the operation, while that of CD4+ lymphocytes transiently increased and then returned to the pre-operative level. Natural killer activity transiently decreased to a slight degree 4 days after the operation and then returned to the pre-operative level 21 days after the operation. Side effects, such as nausea, vomiting and leg discomfort, were seen in the patient (Case 1) treated with carboplatin, but were completely reversible. These results suggest that hyperthermic isolated limb perfusion with concomitant infusion of carboplatin or beta-interferon is effective in suppressing the growth of metastatic malignant melanomas of the lower limb.

Clinical trial of a combination of rice bran fiber and cholestyramine for promotion of fecal excretion of retained polychlorinated dibenzofuran and polychlorinated biphenyl in Yu-Cheng patients.

A clinical trial using the combination of rice bran fiber (RBF) and cholestyramine (CHO) was carried out on Yu-Cheng patients in 1993-1994. By the analysis of blood and stool samples collected from the patients before and after (or during in the case of stool), it was verified that the administration of RBF and CHO is effective for excretion of polychlorinated biphenyl (PCB) (p < 0.05) and polychlorinated dibenzofuran (PCDF), especially 2, 3, 4, 7, 8-pentachlorodibenzofuran (p < 0.05). However, the degree of effectiveness varied upon individual patients from 60 to 160% for 2, 3, 4, 7, 8-pentachlorodibenzofuran, from 30 to 110% for 1, 2, 3, 4, 7, 8-hexachlorodibenzofuran and from 50 to 190% for PCB, respectively.

[Levels of PCDDs, PCDFs and coplanar PCBs in the blood and stool of Taiwanese Yu-Cheng patients].

Levels of PCDDs, PCDFs and coplanar PCBs were measured in the blood and stool obtained from seventeen patients with Yu-Cheng in Taiwan. The average total concentrations of PCDDs, PCDFs and coplanar PCBs in the blood collected from Yu-Cheng patients in January 1993 and August 1994 were 590 and 570 pg Toxic Equivalents (TEQs)/g lipid, respectively. The concentrations of PCDDs, PCDFs and coplanar PCBs in the blood of patients were 21, 540 and 10 pg TEQs/g fat, respectively. On the other hand, the fecal excretion of PCDDs, PCDFs and coplanar PCBs were 26, 720 and 15 pg TEQs/day, respectively. The composition of congeners in the feces was quite similar to the one in the blood. The half lives were estimated as 9.1 and 8.6 years for 2, 3, 4, 7, 8-pentachlorodibenzofuran and 1, 2, 3, 4, 7, 8-hexachlorodibenzofuran based on fecal excretion, respectively.

[Dermatological findings in the annual examination of the patients with Yusho in 1993-1994].

We reported the grades of severity of skin symptoms and the blood PCB patterns and concentrations in Yusho patients who were examined in the annual examinations in 1993 and 1994. The skin severity grades and skin severity scores showed the same tendency as recorded in the last 15 years. Approximately 20% of the patients, however, claimed the tendency of recurrence of skin infections and/or infected epidermal cysts, and still approximately 30% of the patients exhibited acneiform eruptions. Ten patients recorded with more than 6 points of skin severity scoring in these examinations were further investigated their initial skin severity grades and blood PCB patterns and concentrations. It was found that most of their blood PCB patterns showed the A pattern, and the level of the blood PCB in one patient was 55.5 ppb in the initial stage. Five patients out of 10 patients still have more than 10 ppb of blood PCB concentration. Thus, it is concluded that small populations of Yusho patients still have active skin symptoms with considerable blood PCB levels even at present.