Development of an ex vivo breast cancer lung colonization model utilizing a decellularized lung matrix.

The metastatic spread of cancer cells to distant sites represents the major cause of cancer-related deaths in breast cancer patients, and lungs are one of the most common sites for metastatic colonization. Developing a physiologically relevant tissue culture model to mimic lung colonization of breast cancer is crucial for the investigation of the biology of cancer metastasis and evaluation of drug treatment efficacy. Here, we describe an ex vivo lung colonization assay for breast cancer using the native three-dimensional (3D) lung extracellular matrix. The native matrix was isolated from murine lungs using a decellularization technique, and the preservation of extracellular matrix (ECM) composition, integrity and mechanical properties was confirmed. We showed that metastatic MDA-MB 231 and 4T1 cells invaded and colonized in the decellularized lung matrix, whereas only a small mass of non-metastatic MCF7 cells survived under the same condition. Furthermore, knockdown of ZEB1, an epithelial-mesenchymal transition (EMT) inducer, significantly reduced invasion and colonization of MDA-MB 231 cells in the decellularized lung, suggesting an important role of EMT in breast cancer metastasis. We conclude that the decellularized lung retains the biophysical and biochemical properties of the lung ECM and provides a powerful tool to investigate the lung colonization of breast cancer.

Distribution of androstenedione and its effects on total free fatty acids in pregnant rats.

Androstenedione, an anabolic steroid used to enhance athletic performance, was administered in corn oil by gastric intubation once daily in the morning to nonpregnant female rats at a dose of 5 or 60 mg/kg/day, beginning two weeks before mating and continuing through gestation day (GD) 19. On GD 20, the distribution of androstenedione and other steroid metabolites was investigated in the maternal plasma and target organs, including brain and liver. The concentration of estradiol in plasma approached a statistically significant increase after treatment as compared with the controls, whereas the levels of androstenedione, testosterone and progesterone were not significantly different from the controls. In the liver, the concentrations of androstenedione and estradiol only were increased in a dose-related manner. None of these steroids was detectable in the brain. Androstenedione treatment also produced changes in the level of selected free fatty acids (FFAs) in the maternal blood, brain, liver and fetal brain. The concentrations of palmitic acid (16:0) and stearic acid (18:0) in the plasma were not significantly different between the controls and treated rats. However, oleic acid (18:1), linoleic acid (18:2) and docosahexaenoic acid (DHA, 22:6) were 17.94 +/- 2.06 microg/ml, 24.23 +/- 2.42 microg/ml and 4.08 +/- 0.53 microg/ml, respectively, in the controls, and none of these fatty acids was detectable in the treated plasma. On the other hand, palmitic, stearic, oleic, linoleic and DHA were present in both control and treated livers. Among the FFAs in liver, linoleic and DHA were increased 87% and 169%, respectively, over controls. Palmitic, stearic and oleic acids were not significantly affected by the 60 mg/kg treatment. These were present in both control maternal and fetal brains, whereas linoleic acid was found only in fetal brain control. DHA was present only in the control maternal brain (0.02 +/- 0.02 microg/mg protein) and fetal brain (0.24 +/- 0.15 microg/mg protein). The results indicated that androstenedione exhibits significantly different effects on the FFA composition among target organs during pregnancy.

Effects of oral androstenedione on phospholipid fatty acids, ATP, caspase-3, prostaglandin E(2) and C-reactive protein in serum and livers of pregnant and non-pregnant female rats.

Androstenedione, a steroidal dietary supplement taken to enhance athletic performance, could affect serum and liver lipid metabolism, induce liver toxicity or alter inflammatory response depending on dose and duration of exposure. Pregnancy could further exaggerate these effects. To examine this, mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Serum was collected and livers were removed from dams on gestation day 20 and from non-pregnant rats after 5 weeks of treatment. Androstenedione had no effect on serum total cholesterol, triglycerides or HDL-cholesterol, but significantly decreased C-reactive protein in pregnant rats and prostaglandin E(2) in serum of both pregnant and non-pregnant rats. There were treatment related decreases in liver ATP and, to a lesser degree, caspase-3 and no change in alkaline phosphatase of pregnant female rats. Androstenedione decreased docosahexaenoic acid in both serum and liver phospholipids of pregnant female rats. In conclusion, oral androstenedione did not result in overt hepatotoxicity in pregnant female rats, but produced modest changes in lipid metabolism and may impair regeneration of injured hepatic cells or tissue.

Effects of oral androstenedione on steroid metabolism in liver of pregnant and non-pregnant female rats.

It is unknown whether androstenedione, a steroidal dietary supplement taken to enhance athletic performance, can affect physiological hormone levels by altering liver enzyme activities that metabolize steroid hormones. Altered hormone levels could be especially devastating during pregnancy. Mature female rats were gavaged with 0, 5, 30 or 60 mg/kg/day androstenedione beginning two weeks prior to mating and continuing through gestation day 19. Non-pregnant female rats were gavaged over the same time frame with 0 or 60 mg/kg/day androstenedione. Livers were removed from dams on gestation day 20 and from non-pregnant rats after five weeks' treatment. Liver microsomes were incubated with 200 microM testosterone, and the reaction products were isolated and analyzed by HPLC. In pregnant rats, formation of 6alpha-, 15beta-, 7alpha-, 16beta-, and 2beta-hydroxytestosterone was increased significantly vs. control at the highest dose level only. Formation of 6beta-hydroxytestosterone increased significantly at both the 30 and 60 mg/kg/day dose levels. In non-pregnant rats, 60 mg/kg/day androstenedione significantly increased formation of 15beta-, 6beta-, 16beta-, and 2beta-hydroxytestosterone. The data suggest that high oral doses of androstenedione can induce some female rat liver cytochromes P450 that metabolize steroid hormones and that the response to androstenedione does not differ between pregnant and non-pregnant female rats.

Developmental effects of serum from flaxseed-fed rats on cultured rat embryos.

Gestation day 9.5 rat embryos were cultured for 45 h in serum obtained from pregnant rats that had been fed throughout gestation with either a control diet (based on the AIN-93 formulation), a diet supplemented with flaxseed (20% or 40%, w/w), or a diet supplemented with de-fatted flaxseed ("flaxseed meal", 13 or 26%, w/w). The embryos were fixed in neutral formalin at the end of culture. Overall growth and development was assessed, and the presence of abnormalities was noted. A significant inhibition of growth (as determined by crown-rump length) relative to control was observed in embryos cultured in serum from rats fed the 20% flaxseed diet. The incidence of spontaneous heart inversions was increased significantly in the embryos cultured in serum from the 20% flaxseed and 26% flaxseed meal fed rats. The incidence of flexion defects was increased significantly in embryos cultured in serum from 20% flaxseed-fed rats. The lack of an apparent dose response in any of the statistically significant effects suggests that the observed anomalies were chance occurrences unrelated to the treatment group from which serum was obtained. It is therefore concluded that diets high in flaxseed or flaxseed meal do not result in serum factors that are directly embryotoxic to organogenesis-staged rat embryos. This finding is consistent with the findings of a parallel in vivo rat teratology study where no significant embryotoxicity attributable to flaxseed exposure was observed.

Flaxseed increased alpha-linolenic and eicosapentaenoic acid and decreased arachidonic acid in serum and tissues of rat dams and offspring.

The effects of dietary flaxseed (FS), and defatted flaxseed meal (FLM) on serum and tissue fatty acid profiles were investigated. Pregnant Sprague-Dawley rats were fed AIN-93 based diets balanced in calories, fat, nitrogen, and fiber. Diets contained 0, 20%, 40% FS or 13% or 26% FLM by weight. The control, FS and FLM diets differed in linoleic acid to alpha-linolenic acid (ALA) fatty acid ratio. These diets were fed continuously during gestation, suckling period and 8 weeks post-weaning (F(1)). FS fatty acids were bioavailable and metabolized by pregnant and F(1) rats. ALA and eicosapentaenoic acid increased; linoleic and arachidonic acid decreased; and docosahexaeonic acid was unchanged in serum, 'gastric milk' and liver of FS and FLM-fed pregnant and F(1) rats. FS more than FLM, changed fatty acids profiles, but FLM and 40% FS significantly reduced serum cholesterol. Dietary 40% FS may have increased oxidative stress as evidenced by a reduction in liver vitamin E.

Impact of high flaxseed diet on mitogen-induced proliferation, IL-2 production, cell subsets and fatty acid composition of spleen cells from pregnant and F1 generation Sprague-Dawley rats.

Flaxseed (FS) being rich in alpha-linolenic acid may alter the immune parameters. Therefore, we assessed the impact of FS and defatted flaxseed meal (FLM) on fatty acid composition, cell subsets, proliferation and IL-2 production by splenic lymphocytes. Pregnant female Sprague-Dawley rats were fed diets containing 0% FS and FLM, 20 or 40% FS, 13 or 26% FLM during gestation or gestation, lactation and 8 week post-weaning period. FS and FLM resulted in up to 8.3 fold and 4.6 fold increase in splenic ALA among pregnant rats, 4.5 fold and 1.2 fold increase in splenic ALA among F(1) generation rats. Splenic linoleic acid (LA) and arachidonic acid (AA) were 18 and 40% lower in 40% FS fed pregnant rats, and AA was 15% lower in all the other groups. Among F(1) rats, splenic LA and AA were 16 and 48% lower in 40% FS group, and AA was 18% lower in 20% FS and 26% FLM groups. Concanavalin A and phytohemagglutinin mediated proliferation of spleen cells were 60 and 52% lower in 40% FS fed pregnant and F(1) generation rats, respectively. No significant changes were observed in the cell subsets or IL-2 production by splenic cells from different groups.

Role of transurethral biopsy sampling of the prostate to diagnose prostate cancer in men undergoing surgical intervention for benign prostatic hyperplasia.

PURPOSE: Newer minimally invasive surgical procedures are being used to treat men with significant benign prostatic hyperplasia (BPH). These modalities do not allow retrieval of prostate tissue for histologic review. The goal of our study was to assess the value of transurethral biopsies in detecting prostate cancer in men who would undergo surgical intervention for BPH. MATERIALS AND METHODS: Between September 1997 and January 1999, 422 men undergoing transurethral resection of the prostate (TURP) had transurethral biopsies obtained before completing the TURP. Pathology reports as well as prostate-specific antigen (PSA) results were reviewed and analyzed to determine when cancer was present. RESULTS: Pathological examination revealed that cancer was found in 53 men (12.5%). The transurethral biopsies detected cancer in 32 of 53 (60.4%). No cancers were found in the transurethral biopsy specimen only. Of the 21 cancers missed by transurethral biopsy, 7 were stage T1b. PSA level >10 ng/mL increased the likelihood of finding cancer. CONCLUSIONS: Transurethral biopsy sampling is unreliable for detecting prostate cancer in men with clinically significant BPH. Significant cancers are missed if transurethral biopsies are used to determine the presence of carcinoma before minimally invasive surgical therapy for BPH.

Detecting potential teratogenic alkaloids from blue cohosh rhizomes using an in vitro rat embryo culture.

The novel alkaloid thalictroidine (1), as well as the known alkaloids taspine (2), magnoflorine (3), anagyrine (4), baptifoline (5), 5,6-dehydro-alpha-isolupanine (6), alpha-isolupanine (7), lupanine (8), N-methylcytisine (9), and sparteine (10), were identified from an extract of Caulophyllum thalictroides rhizomes. N-Methylcytisine exhibited teratogenic activity in the rat embryo culture (REC), an in vitro method to detect potential teratogens. The structure of 1 was elucidated using various spectroscopic methods, primarily by NMR techniques. Thalictroidine, anagyrine, and alpha-isolupanine were not teratogenic in the REC at tested concentrations. Taspine (2) showed high embryotoxicity, but no teratogenic activity, in the REC.

Effect of peanut oil and randomized peanut oil on cholesterol and oleic acid absorption, transport, and distribution in the lymph of the rat.

Peanut oil was shown to be atherogenic in cholesterol-fed rats, rabbits, and monkeys. However, after randomization, a process in which the fatty acids in peanut oil are randomly rearranged, its atherogenicity was significantly reduced in cholesterol-fed rabbits and monkeys. The mechanism for this effect remains unknown. This study was designed to investigate whether the absorption, transport and distribution of dietary cholesterol and oleic acid in the lymph were altered in the presence of peanut oil or randomized peanut oil. Previous investigators collected lymph through the mesenteric duct for 6 h and analyzed lymph for cholesterol. In the present study, lymph fluids were collected at timed intervals for up to 8 h and then at 24 h via the thoracic duct. Cholesterol and oleic acid (fatty acid) were estimated not only in the whole lymph but also in lymph lipoprotein fractions and in major lipid fractions. A 24-h lymph collection will enhance accuracy as short-term fluctuations in lipid absorption will not affect the results. Thoracic duct lymph collection is quantitative compared to mesenteric duct lymph collection, which provides only a fraction of the total lymph. Rats were given a lipid emulsion containing either peanut oil or randomized peanut oil. The emulsion also contained cholesterol, oleic acid, and sodium taurocholate in saline and was given through a duodenal catheter. Results show that absorption, transport, and distribution of cholesterol and oleic acid in the lymph fluids were similar in both dietary groups. These results suggest that the atherogenicity of peanut oil may be due to other events taking place subsequent to the release of cholesterol-containing chylomicrons and very low density lipoprotein by the small intestinal epithelial cells into the blood or may be due to the triglyceride structure itself.

Assessment of the embryotoxic potential of the total hydrolysis product of fumonisin B1 using cultured organogenesis-staged rat embryos.

Aminopentol (AP1) is the total hydrolysis product of fumonisin B1 (FB1), the major and best characterized of the fumonisins, which are mycotoxins that are common contaminants of corn and corn meal. Some human populations expected to have significant exposure to AP1 have a high incidence of babies born with neural tube defects (NTD). The embryotoxicity of AP1 was evaluated in cultured rat embryos. Gestation day 9.5 embryos were exposed to 0, 3, 10, 30, 100 or 300 microM AP1 throughout the entire 45-hr culture period. At 100 microM AP1, growth and overall development were reduced significantly. There was also a significant increase in the incidence of abnormal embryos. 29% of the embryos had NTD, and 36% of the embryos had other abnormalities. At 300 microM AP1, the incidence of NTD was 15%, and 85% of the embryos had other abnormalities. These findings suggest that AP1, at concentrations of 100 microM and above, can induce NTD in organogenesis-stage cultured rat embryos. However, these NTD are in conjunction with significant overall retardation of growth and development as well as significant increases in the incidence of other defects. These studies also showed, when compared with previous findings, that AP1 is over 100-fold less toxic than FB1 to cultured rat embryos.

Effects of ischemia-hypoxia induced by interruption of uterine blood flow on fetal rat liver and brain enzyme activities and offspring behavior.

The effects of acute perinatal ischemia-hypoxia on fetal liver and brain energy metabolism, fetal brain total free fatty acid concentration and subsequent offspring behavior were investigated in rats. Ischemia-hypoxia was induced at term either by ligation of the uterine blood vessels or submersion of the entire uterine horn in warmed saline. Fetuses of the adjacent horn served as within-dam controls for all assessments and fetuses of dams which had not undergone the surgical stress served as independent controls for enzyme assays. Ischemia-hypoxia was associated with reduced activity of fatty acid synthase in the liver and brain. Total free fatty acid concentration significantly increased in the fetal hypoxic brain. Pups not used for enzyme analyses were cross-fostered for behavioral assessments. Relative to the enzymatic alterations, there were few behavioral alterations associated with ischemia-hypoxia. At postnatal day 30, rats made hypoxic by ligation of the uterine blood vessels had decreased caudate nucleus and brain stem weights relative to within-dam controls. At postnatal day 85, rats made hypoxic by submersion of the uterine horn had decreased olfactory bulb weight. The results of this study indicate an initial acute response to a brief period of ischemia-hypoxia at term pregnancy in the fetal rat brain and liver.

A 54-month evaluation of a popular very low calorie diet program.

BACKGROUND: Thirty-three percent of the adult American population over the age of 20 is obese. Many attempts to treat this increasingly occurring problem have had poor results. Both achieving weight loss and maintaining weight loss are difficult; however, current treatments appear more effective in achieving weight loss than in maintaining weight loss. The current study followed a cohort of patients to analyze weight maintenance and predictors of weight maintenance in a 26-week, formula-based, very low calorie diet program. METHODS: The study population consisted of a consecutive sample of 145 overweight patients who entered a very low calorie diet program and were contacted at 54 months after program entry. RESULTS: For men, the average initial weight loss at program termination was 27.2 kg (22% of original weight) and for women, 19.3 kg (18.8% of original weight). At 54 months after program entry, the average maintained loss was 5.1 kg (4.3% of original weight), at a cost of $630 per kg of long-term weight loss. There was no significant difference in maintained weight loss between men and women. Twenty-six percent of patients maintained a medically significant weight loss of 10% of entry weight. Subjects who exercised regularly maintained an average of 9.6 kg compared with 1.3 kg for nonexercisers. Those who attended the program for a longer period, and exercised more, maintained their weight better. The 54-month weight loss was similar to that seen at 30 months but markedly less than that at 18 months. CONCLUSIONS: Very low calorie diet programs have limited long-term success that may not justify the risk of adverse effects and high costs. Longer program attendance and continued exercise are associated with improved weight maintenance. Evaluation of dietary programs should be based on a sample of consecutive patients followed for a minimum of 2 years after program completion.

Growth patterns in selected organs of the miniature swine as determined by gross macromolecular composition.

As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, the brain, kidney, liver, pancreas, and spleen from male animals were examined for changes in RNA, DNA, and protein contents from 1 to 196 d after birth. Distinct patterns were found for changes with age in macromolecular levels. Protein levels increased from d 1 to 56 in all organs except spleen, in which little change was noted. Gel electrophoresis showed little qualitative change in the liver protein profile during this period. A fat-free, non-nucleic acid, protein-containing fraction, insoluble in hot alkali, appeared in the brain after approximately 1 wk following birth. DNA concentrations decreased markedly from d 1 to d 196 for brain, kidney, and spleen but decreased more gradually for liver and pancreas. RNA levels declined slightly or remained the same in all organs except pancreas, where a large increase occurred from d 1 to weaning (56 d). Growth proceeded in all organs by increases in cell number (hyperplasia), as evidenced by increases in total (level or concentration x organ weight) DNA, or by hypertrophy, as evidenced by increases in the ratio of protein to DNA or by a combination of both processes. Hypertrophic growth was attained by d 56 and continued to sexual maturity in all organs except spleen. Hyperplastic growth continued to sexual maturity in all organs except brain.

Rat embryo culture to detect nutritional deficiency in women with poor reproductive histories.

The cause of habitual early pregnancy loss is not known for most affected couples. It has been proposed that a deficiency of amino acids or other nutrients may contribute to early embryo loss, and an assay based on culture of rat embryos in human serum has been proposed to evaluate women with poor reproductive histories. We tested this assay in women with unexplained infertility (n = 27), habitual abortion (n = 15), and normal midtrimester pregnancies (n = 10) by examining the ability of subject's serum to support the normal development of rat embryos in culture with and without supplemental vitamins and amino acids. Nonpregnant women with nutrient deficiencies identified in this manner were given oral supplements or placebo and were retested. A similar proportion of women in each group had serum that was unable to support the normal development of rat embryos without supplemental vitamins and amino acids. When oral supplements were used, most sera were able to support normal embryo growth. There were no seroconversions on placebo. In spite of the apparent success in producing seroconversions on oral supplementation, only two women conceived, one on the placebo treatment and one on nutritional supplements. Because serum nutrient deficiencies identified by rat embryo culture could not distinguish normal pregnant women from women with unexplained infertility or habitual abortion, and because of the low pregnancy rates, we could not confirm the utility of this assay for the general population of women with habitual abortion.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty-month evaluation of a popular very-low-calorie diet program.

OBJECTIVE: To analyze weight maintenance, cost, and predictors of weight maintenance in a formula-based very-low-calorie diet program. DESIGN AND SETTING: Consecutive sample of patients evaluated at 30 months after program entry at a community hospital in Orange Park, Fla. PATIENTS: Consecutive sample of 306 patients who entered a very-low-calorie diet program. Of these, 255 met inclusion criteria. INTERVENTION: Patients entered a 26-week very-low-calorie diet program. At 30 months after program entry, questionnaires were mailed. Data were collected via telephone interview, as needed. MAIN OUTCOME MEASURES: Initial and maintained weight loss, and association of weight loss to the following factors: insurance coverage, continued exercising, weeks attended, exit weight in relation to ideal weight, and cost per kilogram of weight loss. RESULTS: Medically significant weight loss of 10% was initially achieved by 90% of patients and maintained by 33%. The average initial weight loss was 21.4 kg and the maintained weight loss was 6.5 kg for all patients. For those who remained in the program 19 weeks (61%), the initial weight loss was 25.6 kg and the maintained weight loss was 9.2 kg. Exercisers maintained more than twice as much weight loss as nonexercisers. Men lost a larger percentage of weight (22% vs 19%) and maintained more of that loss (29.5% vs 8.3%). Maintenance was not associated with insurance coverage and at how close patients came to achieving ideal weight. The cost was $396 per kilogram of weight loss maintained. An improved sense of well-being was expressed by 71% of patients. CONCLUSIONS: Very-low-calorie diet programs can be effective in maintaining a medically significant weight loss in some patients at 30 months after program entry. Longer attendance and regular exercise help weight maintenance. The high costs and rate of weight regain indicate the need to find a more affordable and effective strategy for weight loss and maintenance.

Evidence that spontaneous situs inversus in cultured neural plate staged rat embryos is additive with and not mediated through adrenergic mechanisms.

Approximately 50% of untreated presomite rat embryos in culture have demonstrated inversions of cardiac looping (laeval instead of dextral) or tail flexure (left-sided instead of right-sided), or both. This spontaneous situs inversus (SI) was not accompanied by growth inhibition or any other observable defects. The incidence of SI was directly related to the stage at dissection, and all heart defects and most flexure defects were eliminated by delaying explantation to the early somite stage. The incidence of SI was not lowered significantly either by removal of endogenous catecholamines from the culture serum by dialysis or by inclusion of alpha- or beta-adrenergic antagonists in the medium. However, the alpha-adrenergic agonist L-phenylephrine (50 micrograms/ml) increased the incidence of SI to 73%. These findings appear to rule out adrenergic mechanisms as a cause of spontaneous SI in cultured, neural plate-staged rat embryos but suggest a mechanism, yet unknown, that is additive with SI induced by alpha-adrenergic agonists. The low incidence of non-SI-related defects suggests that the high incidence of SI is not an artifact of suboptimal culture conditions. The virtual absence of SI in embryos cultured in bovine serum, a medium in which overall embryonic growth and development were retarded, provides further evidence against nonspecific artifacts.

Methionine and iron as growth factors for rat embryos cultured in canine serum.

Development of headfold-staged rat embryos cultured in canine serum containing various supplements was compared with development in rat serum to seek suitable alternatives to rat serum in rodent embryo culture and to identify nutritional factors for cultured rodent embryos that may have relevance for normal mammalian embryonic growth and development. Supplementation of canine serum with glucose, methionine, and a lipophilic iron chelate allowed growth and development of cultured rat embryos, approximating those obtained with rat serum. These findings suggest that properly supplemented canine serum can serve as a suitable rodent embryo culture medium and that glucose, iron, and methionine may be important nutrients in mammalian embryonic development.

Seafood shucking as an etiology for Aeromonas hydrophila infection.

Aeromonas hydrophila has been increasingly documented as a hardy organism responsible for severe infection in the compromised host. This case report illustrates how A hydrophila may survive prolonged freezing and how seafood shucking may cause sepsis. This report serves as a caution to the immunocompromised seafood lover.

Teratological research using in vitro systems. I. Mammalian whole embryo culture.

Approximately 390 literature references (through spring 1986) were reviewed for mammalian whole embryo culture procedures, with particular attention to the development of those cultures as systems for teratogenicity testing. The existing procedures could be conveniently divided into three groups, which are defined by the periods of embryogenesis that they embrace: preimplantation, peri-implantation, and post-implantation culture systems. The literature on peri-implantation embryo culture was sparse, and it did not appear that this procedure is being actively developed as a teratogen screening test. The extensive literature on both preimplantation and postimplantation embryo culture suggested considerable use of these two methods in evaluating embryotoxicants. The following discussion was compiled from information gleaned from all references. However, in the interest of brevity, only representative articles are specifically cited. Because the background and methodology for each system are distinct, each system will be discussed separately.