Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice.

Previously our laboratory has shown that the soy isoflavone, genistein, stimulates growth of human breast cancer (MCF-7) cells in vivo and in vitro. In this study, the dose-response analysis of genistein at the physiologically achievable concentration range between 125 and 1,000 microg/g in the diet was conducted in ovariectomized athymic nude mice implanted with MCF-7 cells. We hypothesized that genistein at this concentration range can stimulate dose-dependently the breast tumor growth, cell proliferation and an estrogen-responsive pS2 gene induction. Tumor size and body weight were monitored weekly. At completion of the study, we analyzed cellular proliferation of tumors using incorporation of BrdU, pS2 expression of tumors using a Northern blot analysis and total genistein level in plasma using liquid chromatography-isotope dilution mass spectrometry (LC-ES/MS). Dietary genistein (> or = 250 microg/g) increased tumor size in a dose-dependent manner [8.4x the negative control (NC) group in the 250 microg/g group, 12.0x in the 500 microg/g group, 20.2x in the 1,000 microg/g group and 23.2x in the positive control (PC) group]. The percentage of proliferating cells was significantly increased by genistein at and above 250 microg/g (5.3x the NC group in the 250 microg/g, 5.6x in the 500 microg/g, 5.0x in the 1,000 microg/g and 4.8x in the PC group). Expression of pS2 mRNA was also significantly increased with increasing dietary genistein levels (11.25x the NC group in the 500 microg/g group and 15.84x in the 1,000 microg/g group). Total plasma genistein concentrations were between 0.39 and 3.36 micromol/L in mice fed between 125 and 1,000 microg/g genistein. In conclusion, dietary treatment with genistein at physiological concentrations produces blood levels of genistein sufficient to stimulate estrogenic effects, such as breast tumor growth, cellular proliferation and pS2 expression in athymic mice in a dose-responsive manner similar to that seen in vitro.

Dietary genistin stimulates growth of estrogen-dependent breast cancer tumors similar to that observed with genistein.

The estrogenic soy isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo. Genistin is the glycoside form of genistein and the predominant form found in plants. It is generally believed that genistin is metabolized to the aglycone genistein in the lower gut. However, it is unclear if the rate of metabolism of genistin to genistein is sufficient to produce a level of genistein capable of stimulating estrogen-dependent breast cancer cell growth. Our hypothesis was that dietary genistin would stimulate tumor growth similar to that observed with genistein in athymic mice. To test this hypothesis, genistin or genistein was fed to athymic mice containing xenografted estrogen-dependent breast tumors (MCF-7). Mice were fed either genistein at 750 p.p.m. (parts per milllion) or genistin at 1200 p.p.m., which provides equal molar concentrations of aglycone equivalents in both diets. Tumor size was measured weekly for 11 weeks. At completion of the study, half of the animals per treatment group were killed and tumors collected for evaluation of cellular proliferation and estrogen-responsive pS2 gene expression. Incorporation of bromo-deoxyuridine into cellular DNA was utilized as an indicator of cellular proliferation. Dietary genistin resulted in increased tumor growth, pS2 expression and cellular proliferation similar to that observed with genistein. The remaining mice were switched to diets free of genistin and genistein. When mice were placed on isoflavone free diets, tumors regressed over a span of 9 weeks. Next, we examined how effectively and where metabolism of genistin to genistein occurred in the digestive tract. We present evidence that demonstrates conversion of genistin to its aglycone form genistein begins in the mouth and then continues in the small intestine. Both human saliva and the intestinal cell-free extract from mice converted genistin to genistein. In summary, the glycoside genistin, like the aglycone genistein, can stimulate estrogen-dependent breast cancer cell growth in vivo. Removal of genistin or genistein from the diet caused tumors to regress.

Irreducible dislocations of the cervical spine with a prolapsed disc: preliminary results from a treatment technique.

STUDY DESIGN: The preliminary results from a treatment technique for irreducible dislocations of the cervical spine with prolapsed disk are reported. OBJECTIVE: To report the success of a technique for grafting and instrumentation of the anterior cervical spine before reduction. This technique is useful in cervical fracture-dislocations irreducible through the anterior approach that must be approached first from the front because of a prolapsed disc. SUMMARY OF BACKGROUND DATA: In the treatment of cervical facet dislocations, a third anterior procedure often is necessary to accomplish the anterior instrumentation and fusion. The reported technique describes a method that eliminates this third procedure by using a cervical buttress plate. METHODS: Between August of 1996 and October 1998, four patients had dislocation of the cervical spine with a prolapsed disc that could not be reduced using the anterior approach. After discectomy and endplate preparation, a tricortical bone graft was harvested from the iliac crest, placed in the interspace, and held with a buttress plate screwed in two places into the superior vertebral body. The anterior wound then was closed. The posterior elements were exposed and the facets reduced by flexing the neck and posteriorly translating the superior segment. Fluoroscopy was used during the reduction to ensure that the graft was pulled into the interspace, that the screws in the buttress plate did not pull out of the superior vertebral body, and that the reduced graft did not impinge on the spinal cord. A posterior fusion was performed and the posterior wound closed. RESULTS: All the patients had consolidation of both anterior and posterior fusions. No cases of instrument failure occurred, either anteriorly or posteriorly. No cases of neurologic deterioration occurred, and no complications were attributable to the use of this technique. CONCLUSION: The reported technique was used successfully in the treatment of four patients with irreducible dislocations of the cervical spine.

Soy diets containing varying amounts of genistein stimulate growth of estrogen-dependent (MCF-7) tumors in a dose-dependent manner.

We have demonstrated that the isoflavone, genistein, stimulates growth of estrogen-dependent human breast cancer (MCF-7) cells in vivo (C. Y. Hsieh et al., Cancer Res., 58: 3833-3838, 1998). The isoflavones are a group of phytoestrogens that are present in high concentrations in soy. Whether consumption of genistein from soy protein will have similar effects on estrogen-dependent tumor growth as pure genistein has not been investigated in the athymic mouse tumor implant model. Depending on processing, soy protein isolates vary widely in concentrations of genistein. We hypothesize that soy isolates containing different concentrations of genistein will stimulate the growth of estrogen-dependent cells in vivo in a dose-dependent manner. To test this hypothesis we conducted experiments in which these soy protein isolates were fed to athymic mice implanted s.c. with estrogen-dependent tumors. Genistein content (aglycone equivalent) of the soy isolate diets were 15, 150, or 300 ppm. Positive (with 17beta-estradiol pellet implant) and negative (no 17beta-estradiol) control groups received casein-based (isoflavone-free) diets. Tumor size was measured weekly. At completion of the study animals were killed and tumors collected for evaluation of cellular proliferation and estrogen-dependent gene expression. Incorporation of bromodeoxyuridine into cellular DNA was used as an indicator of cell proliferation, and pS2 mRNA was used as an estrogen-responsive gene. Soy protein diets containing varying amounts of genistein increased estrogen-dependent tumor growth in a dose-dependent manner. Cell proliferation was greatest in tumors of animals given estrogen or dietary genistein (150 and 300 ppm). Expression of pS2 was increased in tumors from animals consuming dietary genistein (150 and 300 ppm). Here we present new information that soy protein isolates containing increasing concentrations of genistein stimulate the growth of estrogen-dependent breast cancer cells in vivo in a dose-dependent manner.

Arthritis severity and spirochete burden are determined by serotype in the Borrelia turicatae-mouse model of Lyme disease.

Immunodeficient mice infected with Borrelia turicatae, a relapsing fever agent, have a disorder that resembles disseminated Lyme disease. Two serotypes, A and B, differed in their arthritogenicity in both CB-17 SCID and C3H SCID mice. In CB-17 SCID mice infected with serotype A or B, arthritis was assessed by measurement of tibiotarsal diameter, functional ability on a beam walk test, and microscopic assessment of joint inflammation. Serotype B-infected mice had greater joint swelling, functional disability, and leukocytic infiltration in the joints than serotype A-infected mice. Joint swelling and disability peaked at 2 weeks of infection and then decreased, while leukocyte infiltration in the joints persisted. To investigate the basis for the differences in arthritogenicity of serotypes A and B, spirochete burdens in infected mice were measured by quantitative PCR of spirochete DNA in joints, direct immunofluorescence of spirochetes in joints, and counts of spirochetes in the blood. At 2 weeks of infection there were seven times more spirochetes in the joints of serotype B-infected mice than in those of serotype A-infected mice, measured by both quantitative PCR and direct enumeration. Although serotypes A and B had the same infectivity and growth rate in vivo, serotype B spirochetes were eightfold more abundant in the blood than serotype A spirochetes and produced greater fatality in newborn mice. These findings indicate that differences in disease severity in mice infected with serotype A or B are attributable to differences in the spirochete burden in the joints and blood.

Prognostic value of Ki-67 compared to S-phase fraction in axillary node-negative breast cancer.

The primary purpose of this study was to evaluate the prognostic significance of Ki-67, a cell proliferation-associated antigen, in a large group (n = 674) of axillary node-negative breast cancer cases with long-term follow-up and to correlate Ki-67 antigen expression with S-phase fraction. Ki-67 immunostaining was assessed both semiquantitatively and quantitatively. The statistical analysis focused on agreement between methods of Ki-67 quantification, agreement between Ki-67 and S-phase fraction, associations between Ki-67 and other clinical variables, and prognostic value of Ki-67. There was excellent agreement between the two methods of Ki-67 assessment (Spearman rank correlation, rsp = 0.91; P = 0.0001; n = 674) but only weak correlation between either semiquantitative or quantitative Ki-67 and S-phase fraction (rsp = 0.12 and rsp = 0.15, respectively). Ki-67 (overall median, 2%) was independent of tumor size and modestly related to other measures of tumor aggressiveness. Using a cutpoint of 5% (percentage of tumor cells), cases with high Ki-67 exhibited a significantly shorter disease-free survival (Padj = 0.004). In multivariate analysis, high Ki-67 was associated with a 1.8-fold increased risk of recurrence (P = 0.001). In the subgroup with S-phase data, the adjusted relative risk (hazard ratio, 1.9; P = 0.02) was unchanged by inclusion of S phase in the model. This suggests that Ki-67 provides significant independent prognostic information in addition to that contained in tumor size and S-phase fraction.

Ultrastructure of synovial chondromatosis.

Electron microscopic examination in a case of synovial chondromatosis associated with moderate cytological atypia showed chondrocytes with abundant rough endoplasmic reticulum, prominent Golgi complexes, and peripheral aggregates of glycogen. These findings emphasize the similarity in ultrastructural appearance of chondromatosis, mature hyaline cartilage, and benign cartilaginous tumors and thus support the generally benign nature of synovial chondromatosis.

Primary fenal angiosarcoma: a case report.

A 67 year old white male presented with a two week history of mild hematuria and flank pain. Various radiologic studies demonstrated a vascular mass in the right kidney. At nephrectomy a large renal angiosarcoma with fixation to the liver and multiple pulmonary metastases was found.

Luminol-induced neutrophil chemiluminescence.

Out studies suggest that luminol directly enhances the chemiluminescence of human neutrophils. We show that a significant peak in chemiluminescence production in a particle-free system occurs between 5 and 15 min following exposure of cells to micromolar concentrations of luminol. The response is directly related to dose over a wide rane of luminol concentrations and can be inhibited by superoxide dismutase (90%), catalase (100%) and sodium azide (40%). Evidence is presented which suggests that the effect of luminol eliciting a peak in neutrophil chemiluminescence is mediated within intact cells rather than at the cell membrane. Luminol may produce a peak in chemiluminescence by stimulating very low levels of hexose monophosphate shunt activity and superoxide generation or it may simply amplify light production generated by the production of excited oxygen radicals resulting from surface interactions.

Chemiluminescence by polymorphonuclear leukocytes from patients with active bacterial infection.

Polymorphonuclear leukocytes of 18 patients during 19 episodes of active bacterial infection produced increased chemiluminescence (mean +/- standard error [SE], 56.3 +/- 4.4 X 10(3) cpm) when the production was compared to that of 29 uninfected controls (35.3 +/- 2.4 X 10(3) cpm; P less than 0.01). Chemiluminescence production remained increased with persistent infection but fell to the levels of controls with appropriate therapy. Phagocytic uptake as determined with radiolabeled bacteria was increased, and chemotactic responsiveness was markedly enhanced in the patients (mean index +/- SE, 260 +/- 51) when these responses were compared with those of controls (77 +/- 18). Chemiluminescence and chemotactic activity correlated in the patients with bacterial infection (r = 0.76), but one function did not appear to depend upon the intactness of the other. The ratio of cyclic guanosine 3',5'-phosphate to cyclic adenosine 3',5'-hosphate in the polymorphonuclear leukocytes of patients with infections (mean +/- SE, 0.102 +/- 0.0008) was also significantly higher than in controls (0.067 +/- 0.007). These data indicate that the polymorphonuclear leukocytes of the majority of patients with active bacterial infection are in an activated state both functionally and metabolically.

Influence of phenylbutazone on leukocyte chemiluminescence and function.

The effect of phenylbutazone on human leukocyte chemiluminescence, phagocytosis and intracellular killing of bacteria has been examined. A marked reduction of chemiluminescence and intracellular killing of bacteria was observed. The effect on phagocytosis was less pronounced. High drug concentrations nearly abolished light emission, and concentrations equivalent to those obtained in plasma during therapy caused a 25--30% reduction. The effect occurred within less than 10 minutes. No permanent effect upon resting cells was observed. Phenylbutazone reduced the effect of sodium azide on leukocyte chemiluminescnece, indicating that the drug might also inhibit myeloperoxidase dependent chemiluminescnece. Whether these impairments of leukocyte function also take place in vivo resulting in enhanced susceptibility to infection remains unknown.

Role of antibody and complement in opsonization of group B streptococci.

A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody.

Effect of chemoattractants on chemiluminescence.

Upon ingestion of particulate matter, polymorphonuclear leukocytes produce a chemiluminescence that can be measured in a liquid scintillation counter. In the experiments reported here, the influence of three chemoattractants and three chemotactic modulators upon the chemiluminescence induced by opsonized zymosan was studied. The chemoattractants investigated (including bacterial factor derived from Escherichia coli, the simple peptide formylmethionylalanine, and activated human complement), which initiate directed movement when presented to cells in a concentration gradient, significantly enhanced zymosan-induced chemiluminescence. In the absence of opsonized zymosan, however, they had no effect on the chemiluminescence response. In contrast, the chemotactic modulators studied (including carbamylcholine, phenylephrine, and cyclic guanosine 5'-monophosphate, which are not chemotactic by themselves but can enhance or depress the movement of polymorphonuclear leukocytes initiated by chemoattractants) produced no enhancement of chemiluminescence. Other experiments were carried out in which neutrophils were pretreated with cytochalasin D, a compound that inhibits phagocytosis by interacting with microfilaments. Under these conditions, the chemiluminescence induced by opsonized zymosan was markedly reduced, but the response resulting from the addition of a chemoattractant to the leukocyte/zymosan mixture was not. This indicates that the chemiluminescence in response to chemoattractants is not dependent on phagocytosis per se. Neutrophils were also pretreated with dinitrofluorobenzene, a compound that binds amino groups and can be expected to react with proteins on the cell membrane. In these experiments, the chemiluminescence induced by opsonized zymosan and the pronounced spike of activity produced by the addition of a chemoattractant were completely abolished. These results suggest that the polymorphonuclear leukocyte chemiluminescence response to chemoattractants is mediated by cell surface proteins. Thus, chemoattractants may have a dual role in the acute inflammatory response: (i) the initiation and maintenance of directed cell movement, and (ii) enhancement of metabolic steps mediated at the cell membrane, resulting in microbicidal activity.