Antiangiogenic therapy of established tumors in human skin/severe combined immunodeficiency mouse chimeras by anti-endoglin (CD105) monoclonal antibodies, and synergy between anti-endoglin antibody and cyclophosphamide.

Endoglin (EDG; CD105) is a proliferation-associated cell membrane antigen of endothelial cells and is strongly expressed on the tumor-associated angiogenic vascular endothelium. Furthermore, EDG is essential for angiogenesis and a component of the transforming growth factor (TGF)-beta receptor complex. The present three anti-EDG monoclonal antibodies (mAbs), SN6f, SN6j, and SN6k, react strongly with proliferating human endothelial cells but cross-react very weakly with murine endothelial cells. Analysis of Scatchard plot of direct binding of these mAbs to proliferating human umbilical vein endothelial cells showed equilibrium constants of 8.3 x 10(9), 3.1 x 10(9), and 1.0 x 10(9) liter/mol, respectively, for SN6f, SN6j, and SN6k. These mAbs did not react with MCF-7 human breast cancer cells. To facilitate antiangiogenic tumor therapy by these mAbs in animal models, we used human skin/severe combined immunodeficiency (SCID) mouse chimeras bearing tumors of MCF-7. Blood vessels in the chimeras were analyzed by immunostaining with species (human or mouse)-specific anti-CD31 and anti-EDG mAbs including an antihuman EDG mAb termed SN6h. Blood vessels in the completely healed grafted human skins consisted of a mixture of human (43.5%) and murine (56.5%) vessels, whereas only murine vessels were detected in the adjacent murine skins and s.c. tissues. Therefore, murine vessels infiltrate into the human skin grafts from the adjacent murine tissues, whereas the growth of human vessels is limited within the boundary of human skins. Growth of human MCF-7 tumors in the human skin grafts increased the ratio of human:murine vessels. Analyses of the grafted skins before and after tumor transplantation showed that SN6h reacted with tumor-induced angiogenic blood vessels but not with nonangiogenic vessels, whereas antihuman CD31 mAb reacted with both angiogenic and nonangiogenic vessels. The results show that SN6h is capable of distinguishing the tumor-induced angiogenic vasculature from the nonangiogenic vasculature in the present model. Antiangiogenic therapy of the chimeras bearing established MCF-7 tumors was carried out by i.v. administration of a mAb(s) via the tail vein of mice. SN6j and SN6k were effective for suppressing the established tumors, whereas tumor suppression was weaker with SN6f. The results indicate an absence of a direct correlation between antigen-binding avidity and in vivo antitumor efficacy of anti-EDG mAbs and suggest the importance of other factors (e.g., epitopes) in antitumor efficacy. No significant toxicity of the mAbs was detected. Combination of SN6f and SN6k that define mutually nonoverlapping epitopes showed an additive antitumor effect. Combination of SN6j and cyclophosphamide using an antiangiogenic schedule of drug dosing showed synergistic antitumor efficacy. The combination therapy induced lasting complete regression of the established tumors in two of the eight treated chimeras. We examined human and murine blood vessels in large human tumors from the chimeras at the end of therapeutic experiment. The test showed that SN6j therapy resulted in complete suppression of human vessels in the tumors but resulted in only weak suppression of murine vessels. Cyclophosphamide was not effective for suppressing human vessels and only weakly suppressive against murine vessels. Combination of SN6j and cyclophosphamide was effective for completely suppressing human vessels and also effective for partial (i.e., 35%) suppression of murine vessels. The results show that systemic administration of naked antihuman EDG mAbs can suppress established tumors, and the efficacy is markedly enhanced by combining a chemotherapeutic drug using an antiangiogenic schedule of drug dosing. These mAbs should show stronger antitumor efficacy in patients whose tumors depend entirely on human blood vessels.

Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli.

The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems. CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane. In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP. Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E. coli. The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E. coli genome (50%). The introduction of a recombinant plasmid containing the cof gene cluster into the E. coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2. This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.

Association of serum endoglin with metastasis in patients with colorectal, breast, and other solid tumors, and suppressive effect of chemotherapy on the serum endoglin.

In this report, we present data indicating that the increased serum endoglin (EDG; CD105) quantitated by a double-antibody sandwich assay is associated with metastasis in patients with solid tumors including colorectal and breast carcinomas. In addition, we show that chemotherapy exerts a suppressive effect on the serum EDG. EDG is a proliferation-associated cell membrane antigen of human vascular endothelial cells. Furthermore, EDG is essential for angiogenesis. We generated two anti-EDG monoclonal antibodies (mAbs), termed SN6a and SN6h, defining different epitopes of EDG and developed a double-antibody sandwich assay to quantitate serum EDG in patients with solid tumors. SN6h possesses an exceedingly high antigen-binding avidity (K, 1.38 x 10(11) liters/mol), whereas SN6a possesses an ordinary avidity for a mAb directed to a cell surface antigen (K, 2.85 x 10(8) liters/mol). We measured serum samples from 101 patients with solid tumors (34 colorectal cancers, 16 breast cancers, and 51 other cancers), 8 patients with benign diseases, and 31 healthy volunteers. The serum level of EDG was significantly elevated in the patients with metastatic cancers. The mean serum EDG in the 42 metastasis-negative patients was 34.0 +/- 26.8 ng/ml (median value, 27.9 ng/ml), whereas the value in the 59 metastasis-positive patients was 63.8 +/- 72.5 ng/ml (median value, 37.2 ng/ml). The difference in EDG levels between the two groups was statistically significant (P = 0.012). Of the colorectal cancer patients, the difference in EDG levels between the 19 metastasis-negative patients and the 15 metastasis-positive patients was statistically significant (P = 0.02). In addition, the difference between the normal control (n = 31) and the 15 metastasis-positive colorectal cancer patients was statistically significant (P = 0.04). Of the breast cancer patients, the difference in EDG levels between the 11 metastasis-positive patients and the normal control was statistically significant (P < 0.005). In additional studies, we found that chemotherapy suppressed serum EDG levels in cancer patients. Of the 54 metastasis-positive patients with solid tumors, the mean serum EDG in the 32 chemotherapy-receiving [chemotherapy(+)] patients was 44.7 +/- 41.9 ng/ml (median value, 36.1 ng/ml), whereas the value in the 22 chemotherapy(-) patients was 102.4 +/- 99.5 ng/ml (median value, 64.8 ng/ml). The difference in serum EDG between the two groups is statistically significant (P < 0.005). In the majority of metastasis-positive patients who were not receiving chemotherapy, serum EDG was elevated. The results suggest that serum EDG may be a useful marker for monitoring early signs of metastasis and cancer relapse in a long-term follow-up of solid tumor patients.

Angiogenesis and metastasis marker of human tumors.

Tumor growth and metastasis are dependent on angiogenesis. Therefore, certain angiogenesis markers may be useful as metastasis markers and/or the targets for antiangiogenic therapy. We and others have been studying endoglin(EDG; CD105) for such purposes. EDG is a proliferation-associated antigen of endothelial cells and essential for angiogenesis. In addition, EDG is a component of the transforming growth factor(TGF)-beta receptor complex. Expression of EDG is up-regulated in tumor-associated angiogenic vasculature compared with normal tissue vasculature. Microvessel density detected for EDG expression in breast cancer tissues showed a statistically significant correlation with overall and disease-free survival. In addition, elevated serum EDG was associated with metastasis in patients with colorectal, breast, and other solid tumors. On the other hand, We have been targeting EDG on tumor vasculature to suppress tumor growth and metastasis by systemic(i.v.) administration of anti-EDG monoclonal antibodies(mAbs) and immunoconjugates(IMCs). To thid end, we have been using three animal models, i.e., severe combined immunodeficient(SCID) mouse model of MCF-7 human breast cancer, human skin/SCID mouse chimera model bearing MCF-7 tumor, and syngeneic metastasis model of colon-26 adenocarcinoma cells in BALB/c mice. In addition, antiangiogenic activities of anti-EDG mAbs and IMCs were evaluated in mice using the dorsal air sac assay. The IMCs were prepared by coupling deglycosylated ricin A-chain or 125I to individual anti-EDG mAbs. These anti-EDG IMCs and mAbs showed substantial antitumor efficacy and antimetastatic activities without showing severe toxicity. Recently, we generated a recombinant human/mouse chimeric anti-EDG mAb to facilitate clinical application of the mAb.

Selective augmentations of intratumoral 5-fluorouracil concentration by local immunotherapy with OK-432 and fibrinogen.

PURPOSE: Pyrimidine nucleoside phosphorylase is an enzyme that converts 5'-deoxy-5-fluorouridine into its active metabolite, 5-fluorouracil. In colorectal cancer tissue pyrimidine nucleoside phosphorylase has been proven to be produced by macrophages in the cancer stroma despite presence of the cancer cells. We reported that local immunotherapy with OK-432 and fibrinogen induced aggregation of macrophages in the cancer stroma and enforced their pyrimidine nucleoside phosphorylase expression. Thus it was hypothesized that if colon cancer were treated with 5'-deoxy-5-fluorouridine, the 5-fluorouracil concentration in cancer tissues would be enhanced by local immunotherapy. The present study was conducted to investigate whether local immunotherapy for colon cancer could increase the intratumoral 5-fluorouracil concentration in patients given chemotherapy with 5'-deoxy-5-fluorouridine. METHODS: Twenty patients with resectable colorectal cancer were examined in this study. They were given 5'-deoxy-5-fluorouridine (600 mg/day) orally for seven days preoperatively. Nine randomly selected patients underwent intratumoral injection of OK-432 mixed with fibrinogen, which was performed on the third preoperative day (OK-432 and fibrinogen plus 5'-deoxy-5-fluorouridine group); eleven patients were given oral 5'-deoxy-5-fluorouridine only (5'deoxy-5-fluorouridine group). The 5-fluorouracil concentration in tumor tissue and normal colon mucosa tissue was measured, and the influence of the local immunotherapy was assessed. RESULTS: The 5-fluorouracil concentration in the cancer tissue was increased by the local immunotherapy, whereas that in the normal colon mucosa was not influenced. Thus, the influence of local immunotherapy was selective to the cancer tissue where the mixture of OK-432 and fibrinogen was injected. CONCLUSION: In patients with colorectal cancer, selective high 5-fluorouracil concentration in the cancer tissue could be achieved by a combination of 5'-deoxy-5-fluorouridine and local immunotherapy with a mixture of OK-432 and fibrinogen.

[PyNPase expression and cancer progression in the colorectum].

We analyzed PyNPase expression discriminating between cancer and tumor stroma of the colorectum by Western blotting using a newly developed extraction method from microdissected tissue sections fixed with buffered formalin. Analysis of 98 colorectal cancers revealed that PyNPase was as high as 70.2 +/- 18.5 unit/mg protein in the stroma fraction (SF), whereas it was 45.1 +/- 10.5 in the cancer fraction (CF) (p < 0.0001). Vessel density was correlated with PyNPase in the SF but not in the CF. In stage IIIb, 11 cases expressing a high level of PyNPase in the CF showed poorer prognosis than 10 cases with low-level PyNPase expression (p < 0.05), although the level of PyNPase expression in the SF did not affected the patients prognosis. Immunohistochemical examination indicated that PyNPase in the SF was mainly produced by macrophages (M phi), and therefore we investigated the profile of PyNPase production by M phi. In in vitro experiments PyNPase production by M phi was greatly enhanced by stimulation with OK-432, and the culture supernatant had the ability to convert 5'DFUR to 5-FU.

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

PyNPase expression in human colon cancer.

Although colorectal cancer tissue is rich in pyrimidine nucleoside phosphorylase (PyNPase), there is no consensus as to whether cancer cells or stromal cells predominately express PyNPase. We micro-dissected OCT compound embedded frozen tissue sections into epithelial and stromal components and then analyzed the extracted samples separately. The PyNPase expression level was higher in stromal cells than in cancer cells and the difference increased with inflammation induced by the immunostimulator OK432. These results suggest that stromal cells are the major PyNPase source in colorectal cancer.

Analysis of cytotoxic activity of the CD4+ T lymphocytes generated by local immunotherapy.

We previously reported that the anti-tumour effect of OK-432 is considerably enhanced by its intratumoral injection together with fibrinogen. In the present study, we generated killer T cells by culturing tumour-infiltrating lymphocytes from thyroid cancer patients who had received this local immunotherapy. Phenotypic analysis revealed that the T cells were positive for CD3+, CD4+, Leu8-, CD45RO+ and T-cell receptor (TCR)alpha beta+, as well as showing strong surface expression of HLA-DR, CD25, LFA-1 and ICAM-1. The generated CD4+ T cells secreted interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, TNF-beta, and interleukin (IL)-6 (but not IL-4), and exhibited a high level of cytolytic activity against several tumour cell lines. The cytolytic activity of these T cells for Daudi cells was inhibited by preincubation with an anti-intercellular adhesion molecule (ICAM)-1 antibody, but not by preincubation with anti-TCR alpha beta, anti-CD2, or anti-LFA-1 antibodies. Pretreatment with anti-ICAM-1 antibody inhibited T-cell cytolytic activity, but not conjugation with target cells. In addition, incubation with immobilised anti-ICAM-1 enhanced the secretion of IFN-gamma by T cells. We conclude that ICAM-1 expressed on the effector cytotoxic CD4+ T lymphocytes delivers regulatory signals that enhance IFN-gamma secretion.

Abnormalities among offspring of oral and nonoral contraceptive users.

An analysis of 223 children born between 1961 and 1965 to 5,465 women in a controlled experiment at the University of Puerto Rico School of Medicine, regarding the use of oral and nonoral contraceptives, failed to reveal any statistical differences in birth weight and physical abnormalities between the two contraceptive groups. The mean birth weight for the oral group was 7 pounds, 4.1 ounces, whereas for the nonoral group it was 7 pounds, 5.4 ounces. Abnormal physical findings were 10.7% for the oral group and 16.7% for the nonoral group. A comparison is also made between the two groups with respect to incidence of abnormalities by socioeconomic class and by sex of the child, as well as by the number of pregnancies and age of the mother. No significant differences were noted between the groups as an indication of the effect of oral contraceptives.

Changing patterns in cervical cytology among oral and non-oral contraceptive users.

PIP: 9634 patients (21-39 years, with at least 1 normal pregnancy) with no previous experience with oral contraceptives or IUDs, seen at clinics in Rio Piedras, Caguas, and Ponce, Puerto Rico from July 1961 to October 1969 to study their changing patterns in cervical cytology were divided randomly into 2 groups, of which 4846 were given oral contraceptive, Enovid, and 4788 provided with a vaginal contraceptive excluding IUDs, and followed for a period of 6 months-8 years. Separate Pap smears were obtained at admittance to the study by an endocervical aspiration with the pipette and cervical sample with Ayre spatula scraped over the cervico-columnar junction. The cervix was diagrammed showing any erosion. Schiller tests were done. Possible presence of Trichomonas vaginalis was treated. Repeat smears were done at 3-month intervals. 2129 oral patients and 2249 nonoral patients experienced either regression or progression in cytological patterns throughout the 8 years of observation. The analysis is divided into 3 groups, irrespective of the time at which they were observed. Of the 2129 oral patients showing a first change, 55.5% progressed to a higher Pap class, 44.5% regressed to a lower Pap class. In the nonoral group, 55.2% progressed and 44.4% regressed. 1255 patients had a second change: in the oral group, 37.5% progressed, 62.5% regressed. In the nonoral group, 36.7% progressed, 63.3% regressed out of 1258 patients. 690 oral patients showed a third change: 46.1% progressed and 53.9% regressed. Of the 718 nonoral patients 47.1% progressed and 52.9% regressed. There was no significant difference in the pattern of progression and regression between oral and nonoral groups. Oral contraceptives do not aggravate the conditions which lead to development of premalignant lesions in the uterine cervix.^ieng

Thyroid status in long-term, high-dose oral contraceptive users.

PIP: The effect of high-dose, long-term oral contraceptive use on thyroid function and thyroid disease was investigated. Between 1956 and 1962 there were 836 patients treated with Enovid. Initially doses of 10 mg were given, then 5 mg, and finally 2.5 mg. 53 patients used oral contraceptives continuously through a total of 8111 cycles with an average of 156 cycles and an average dose of 3.75 mg/day/cycle. These 53 women were studied for protein bound iodine (PBI), resin triiodothyronine (T3) uptake, total thyroxine (TT), free thyroxine level (FT), radioiodine uptake, and a thyroid scan. Fractional uptakes were done on some. The PBI, T3, TT, and FT tests were done at the Bio-Science Laboratories in California, the others at a local laboratory. Uptake values were abnormally low in 40% of patients. This finding had not been previously reported. Thyroid scannings for 48 patients showed 44 were normal. In 3 patients the thyroid appeared uniformly enlarged but this was not apparent on physical examination. A small cold area was noted in 1 thyroid. No nodule or other variation was found on physical examination. A combination of the PBI test and T3 would be most helpful in evaluating hypo or hyperthyroidism. In the presence of a nontoxic goiter the PBI would be normal or high and the T3 normal or low. Though individual tests showed alterations no definite e vidence of thyroid disease was found in the patients studied.^ieng