[Effects of Peroxisome Proliferator-Activated Receptor γ on Modulating Angiopoietin-Like Protein 4 Synthesis in Caco-2 Cells Exposed to Clostridium butyricum].

Angiopoietin-like protein 4 (ANGPTL4) is considered to be one of the important circulating mediators linking intestinal microorganisms and host lipid metabolism. The objective of this study was to assess the effects of peroxisome proliferator-activated receptor у (PPARγ) on modulating ANGPTL4 synthesis in Caco-2 cells exposed to Clostridium butyricum. The viability of Caco-2 cells and the expression of PPARγ and ANGPTL4 in Caco-2 cells were detected after the Caco-2 cells were co-cultured with C. butyricum at the concentration of 1 x 10^(6), 1 x 10^(7) and 1 x 10^(8) CFU/mL. The results showed that cell viability was enhanced by C. butyricum. Besides, PPARγ and ANGPTL4 expression and secretion in Caco-2 cells was significantly increased by 1 x 10^(7) and 1 x 10^(8) CFU/mL of C. butyricum. Furthermore, the effects of PPARγ on modulating ANGPTL4 synthesis in Caco-2 cells regulated by 1 x 10^(8) CFU/mL of C. butyricum was also be expounded in PPARγ activation/inhibition model based on Caco-2 cells and via ChIP technique. It was found that C. butyricum promoted the binding of PPARγ to the PPAR binding site (chr19: 8362157-8362357, located upstream of the transcriptional start site of angptl4) of the angptl4 gene in Caco-2 cells. However, the PPARγ was not the only way for C. butyricum to stimulate ANGPTL4 production. Taken together, PPARγ played a role in the regulation of ANGPTL4 synthesis by C. butyricum in Caco-2 cells.

Safety evaluation of phytosterols in laying hens: effects on laying performance, clinical blood parameters, and organ development.

Phytosterols are intended for use as a novel food ingredient with plasma cholesterol-lowering activity. Although phytosterols are naturally present in the normal diet, daily consumption is insufficient to ensure plasma cholesterol-lowering levels. Therefore, phytosterols may be added to the diets to achieve the desired cholesterol-lowering activity. A subchronic laying hen safety study was conducted to examine if high-dose phytosterols could affect the safety of hens. Three hundred sixty 21-wk-old Hy-Line Brown laying hens were randomly assigned to 5 groups with 6 replicates of 12 birds each; after 3 wk, birds were fed diets supplemented with 0, 20, 80, 400, and 800 mg/kg of phytosterols for 12 wk. Throughout the study, clinical observations and laying performance were measured. At the end of the study, birds were subjected to a full postmortem examination: blood samples were taken for clinical pathology, selected organs were weighed, and specified tissues were taken for subsequent histological examination. No treatment-related changes that were considered to be of toxicological significance were observed. Therefore, a nominal phytosterol concentration of 800 mg/kg was considered to be the no-observed-adverse-effect level.

Effects of stocking density on growth performance, carcass yield, and immune status of a local chicken breed.

An experiment was conducted to evaluate the effect of stocking density on growth performance, carcass yield, and immune status of a local chicken breed. In total, 840 one-day-old male Suqin yellow chickens were placed into 4-m(2) cages in groups of 50 (low), 70 (medium), or 90 (high) birds. Each treatment was represented by 4 replicates (cages). The cages measured 2.84 × 1.42 m; half of the area of the cage (2 m(2)) was used from 1 to 28 d and the whole cage was used from 29 to 42 d. Stocking densities were 25, 35, and 45 birds/m(2) from 1 to 28 d and 12.5, 17.5, and 22.5 birds/m(2) from 29 to 42 d (low, medium, and high, respectively). Final production (live bird mass after fasting) per unit area was 14.46, 19.46, and 24.23 kg/m(2), respectively, at 42 d of age. Several immune parameters were evaluated, and the growth performance, carcass yield, and meat quality were determined. Body weight at 28 and 42 d of age was significantly reduced as the stocking density increased (P < 0.05). A depression in daily weight gain was noticed from 1 to 28 d and 1 to 42 d of age, and daily feed intake decreased significantly in each period as density increased (P < 0.05). The feed/gain from 29 to 42 d and from 1 to 42 d of age decreased as density increased (P < 0.05). At 42 d, there was no effect of the stocking density on carcass, eviscerated carcass, breast, and abdominal fat yields (P > 0.05). The thigh yield of chickens in the medium-density group improved significantly (P < 0.05) compared with those of the other 2 groups. The water-loss rate, shear force, and meat color of the muscle were unaffected (P > 0.05) by the stocking density, but pH values increased slightly as density increased. No significant difference was noted in the immunological parameters, but the blood total protein and potassium were significantly affected by stocking density (P < 0.05). The findings of this study suggest that increasing the stocking density advantageously affected feed/gain and decreased the final BW, whereas no evidence was found that stocking density caused changes in any of the measured immune parameters.

Effects of rice husk diluted dietary switching on the phenotypic change of gastrointestinal tract in adult ganders.

1. An experiment was conducted to test the directionality, scaling and reversibility of phenotypic responses of the gastrointestinal tract (GIT) of adult ganders to rice husk (RH) diluted dietary switching. 2. A total of 96 140-d-old ganders were acclimatised to a basal diet for 2 weeks. The birds were randomly assigned to 4 treatments. On d 1, diets in the experimental groups were switched from the basal diet to diets which contained 200, 400 or 600 g/kg RH by mass, with no RH in the basal diet. After 21 d, the diet of all the experimental birds was switched back to the basal diet until d 42. 3. Increasing RH content significantly increased feed intake, and a decreased trend appeared after diet-switching. The weights of geese fed on the 600 g/kg RH diet for 21 d reduced, and were significantly less than those of the other three groups, while body weights (BW) of the geese in all groups increased after diet-switching back to the basal diet. At d 21, significantly heavier relative weights of proventriculus, gizzard and all gut components, except duodenum, were observed in birds fed on a 600 g/kg RH diet, and significantly heavier relative weights of gizzard were observed in birds given a 400 g/kg RH diet. Thickness of the two gastric walls, gizzard length and all gut components lengths increased significantly in birds given a 600 g/kg RH diet compared with the other three groups. At d 42, no significant differences were noted in the relative weights or lengths of GIT, except for the caeca, which were significantly heavier in birds fed on 600 g/kg RH diet. 4. The results of the experiment were in accordance with the predictions of the hypothesis that there is matching between loads and capacities. The observed phenotypic responses were directional and scaled to the demands.

Dietary supplementation of methionine mitigates oxidative stress in broilers under high stocking density.

We aimed to investigate whether dietary supplementation of methionine could mitigate intestinal oxidative injury in broilers under high stocking density (HSD). In the grower phase (d 22-42), 576 broilers with similar body weight were randomly chosen and divided into 8 groups in a 2 × 4 factorial experiment. Two different stocking densities (14 and 20 broilers per m2) were tested with 4 different methionine levels: 0.35%, 0.4%, 0.45%, or 0.5%. Intestinal morphological and oxidative stress markers were assessed at the end of the test period. The results showed that mortality of broilers was significantly higher in the HSD group fed 0.35% methionine diet than the other groups, which was reversed by supplementation with 0.40% to 0.50% methionine. HSD significantly decreased feed intake and daily weight gain. HSD treatment significantly decreased T-AOC, activity of GPX (P < 0.01) and increased the level of PCO (P < 0.01), MDA (P = 0.052) of plasma. The decreased glutathione peroxidase activity in the liver and jejunum caused by HSD was alleviated by additional methionine. Supplementation of methionine increased the ration of GSH/GSSG in the plasma. The jejunum villus height and ratio of villus height to crypt depth under low stocking density conditions with 0.40% methionine diet were the highest, whereas the 0.45% methionine group was the highest under HSD conditions. Thus, additional dietary supplementation of methionine mitigates oxidative stress in broilers under HSD conditions and 0.40% to 0.45% methionine can be applied in cage rearing broiler production for amelioration of oxidative stress caused by HSD.

Response of growing goslings to dietary methionine from 28 to 70 days of age.

1. A dose-response experiment with 5 dietary methionine concentrations (23, 33, 43, 53 and 63 g/kg) was conducted with goslings to estimate the growth performance and carcase quality response of growing goslings to dietary methionine from 28 to 70 d of age. 2. A total of 150, 28-d-old birds were randomly distributed to 15 pens with 10 birds per pen according to similar pen weight. There were 5 dietary treatments, each containing three replicate pens. Weight gain, feed intake and feed/gain of goslings from each pen were measured at 2-week intervals from 28 to 70 d of age. At 70 d of age, 4 goslings were selected randomly from each pen and slaughtered to evaluate carcase quality. 3. Significant effects of dietary methionine on daily weight gain (28-56 d) and daily feed intake were determined. Daily weight gain from 28 to 42 d and 28 to 56 d, daily feed intake and gain/feed showed significant quadratic response to increasing dietary methionine, while abdominal fat proportion showed a significant linear response. 4. When dietary CP concentration was 1582 g/kg, the optimal methionine concentrations for growing goslings from 28 to 42 d and 28 to 56 d of age for maximum daily weight gain were 407 and 4.14 g/kg, respectively.

A standard technique with potential diagnostic value for estimating the quadriceps muscle strength of critically ill patients.

OBJECTIVE: Intensive care unit-acquired weakness (ICUAW) is common, and so far, there is no digital technology with a standard procedure to estimate the muscle strength of these patients. Quadriceps maximal isometric voluntary contraction (QMVC) is a precise and reliable procedure to detect quadriceps muscle strength. Therefore, this research aimed to explore whether QMVC measurements can be used in critically ill patients at the bedside as a potential diagnostic method. PATIENTS AND METHODS: Tailor-made computerized equipment was designed to measure the QMVC of critically ill patients at the bedside, following a standard procedure. A total of 22 critically ill patients and 22 age- and sex-matched healthy subjects were divided into group 1 and group 2, respectively. SPASS 21.0 (IBM, Armonk, NY, USA) software was used to analyze the data. RESULTS: All subjects showed good endurance with the QMVC measurements and there were no side effects among these subjects. There was a significant decline in QMVC between group 1 and group 2 (p=0.000). QMVC was correlated closely with the APACHE II Score in group 1 (Pearson correlation, r=-0.427, p=0.047). Among the 10 patients with a Medical Research Council sum score (MRC SS) less than 60 in group 1, it was also correlated closely with the MRC SS (Pearson correlation, r=0.837, p=0.003). CONCLUSIONS: This study describes a standard technique for quantifying quadriceps muscle strength that is feasible for use with critical patients. QMVC can accurately detect the decline of quadriceps muscle strength of critical patients, and it may also decline with the severity of the disease. In the future, this technique might be a potential diagnostic tool for ICUAW.

Effect of a free-range raising system on growth performance, carcass yield, and meat quality of slow-growing chicken.

Experiments were conducted to evaluate the effect of free-range raising systems on growth performance, carcass yield, and meat quality of slow-growing chickens. Slow-growing female chickens, Gushi chickens, were selected as the experimental birds. Two hundred 1-d-old female chicks were raised in a pen for 35 d. On d 36, ninety healthy birds, with similar BW (353.7+/-32.1g), were selected and randomly assigned to 2 treatments (indoor treatment and free-range treatment, P>0.05). Each treatment was represented by 3 groups containing 15 birds (45 birds per treatment). During the indoor treatment, the chickens were raised in floor pens in a conventional poultry research house (7 birds/m2). In the free-range treatment, the chickens were housed in a similar indoor house (7 birds/m2); in addition, they also had a free-range grass paddock (1 bird/m2). All birds were provided with the same starter and finisher diets and were raised for 112 d. Results showed that the BW and weight gain of the chickens in the free-range treatment were much lower than that of the chickens in the indoor floor treatments (P<0.05). There was no effect of the free-range raising system on eviscerated carcass, breast, thigh, and wing yield (P>0.05). However, the abdominal fat yield and tibia strength (P<0.05) significantly declined. The nutrient composition (water, protein, and fat), water-holding capacity, shear force, and pH of the muscle were largely unaffected (P>0.05) by the free-range raising system. The data indicated that the free-range raising system could significantly reduce growth performance, abdominal fat, and tibia strength, but with no effect on carcass traits and meat quality in slow-growing chickens.

Genistein increases progesterone secretion by elevating related enzymes in chicken granulosa cells.

Genistein, a biologically active isoflavone, exists in many soy products. It is well known that genistein binds to both oestrogen receptor alpha (ERα) and oestrogen receptor beta (ERß), but it has a higher affinity to ERß. Genistein can also bind to the G protein-coupled receptor 30 (GPR30, also known as G protein-coupled oestrogen receptor 1 or GPER). Furthermore, weak oestrogenic activity has been found in genistein, but the mechanism of action remains unknown. The aim of this study was to investigate the in vitro effects of genistein on the secretion of progesterone (P4) and oestradiol (E2) in chicken granulosa cells harvested from follicles, as well as the mRNA expression of ERs in these cells. In addition, we examined the expression of key enzymes including steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) in the process of P4 synthesis. The results showed that genistein did not affect the viability of granulosa cells, nor was the proliferating cell nuclear antigen (PCNA) protein changed. Among the 1-, 10-, 100-, and 1,000-nM concentrations tested, treatment with 1 nM genistein for 48 h significantly increased P4 but did not affect E2 secretion. Real-time PCR results showed that the ERß gene expression in granulosa cells was markedly upregulated by 1 nM genistein treatment for 48 h, but there was no significant difference in ERα and GPR30 expression. Genistein also increased the gene expression of StAR, P450scc and 3ß-HSD in the cultured granulosa cells. These results indicate that genistein acts directly on chicken granulosa cells to increase P4 production by upregulating the gene expression of key enzymes through binding in ERß. It may exert positive effects on the reproduction of late-laying hens and act as an effective and safe feed additive for animals.

A mixture of daidzein and Chinese herbs increases egg production and eggshell strength as well as blood plasma Ca, P, antioxidative enzymes, and luteinizing hormone levels in post-peak, brown laying hens.

Dietary supplementation with the isoflavone, daidzein, has been shown to improve egg production in poultry. Additionally, providing Chinese herbs (CH) in the broiler diet has led to increased antioxidative enzyme activity. However, the combined effect of these dietary supplements on hen performance has not been examined. Therefore, the objective of this study was to determine if dietary supplementation with a mixture of daidzein and CH would alter laying performance, egg quality, and blood plasma constituents of post-peak laying hens. At 59 wk of age, Hyline brown hens (240) were randomly allocated to 2 dietary groups and fed for 16 wk. The control group received the basal diet, and a treatment group was fed the basal diet that contained 0.02% of a mixture of daidzein and CH. Egg production and weight were recorded daily and egg quality data were collected at 75 wk of age. Blood plasma antioxidant activity, hormone levels, mineral (Ca and P) content, and osteocalcin content were determined at the end of the study. The results showed that laying rate, egg mass, and shell strength were greater in the daidzein-CH mixture group than the controls (P < 0.05). The plasma glutathione peroxidase, superoxide dismutase, and luteinizing hormone levels were also greater in the daidzein-CH mixture group compared with the control group (P < 0.05). The results of this study reveal that supplementing diets with a daidzein-CH mixture can improve laying performance perhaps by increasing plasma antioxidant activity, luteinizing hormone levels, and mineral content.

The dietary combination of essential oils and organic acids reduces Salmonella enteritidis in challenged chicks.

This study was conducted to determine the effects of essential oils and organic acids (EOA) on Salmonella Enteritidis (S. Enteritidis) challenged chickens. One-day-old specific pathogen-free (SPF) chicks (250) were randomly assigned to 5 groups, with 50 birds in each group. The treatment groups were as follows: 1) basal diet, negative control group (NC); 2) basal diet + S. Enteritidis, positive control group (PC); 3) PC + 4,000 g/t of enrofloxacin (5%), antibiotic group (ENR); 4) PC + 800 g/t of EOA1, thymol-benzoic acid group (TBA); and 5) PC + 800 g/t of EOA2, cinnamylaldehyde-caproic acid group (CCA). At 7 D of age, each bird, except those in NC, was orally gavaged with 0.4 mL of a suspension of 4.4 × 109 cfu S. Enteritidis/mL. Results revealed that ENR reduced bacterial counts in the liver and spleen on days 3, 5, and 7 post-challenge more (P < 0.05) than any other treatments. However, bacterial counts in cecal contents among ENR, TBA, and CCA were similar at 5 and 7 D post-challenge but lower than those of PC. Additionally, the bacterial counts in liver, spleen, and cecum contents in TBA were lower (P < 0.05) than in PC at 3, 5, and 7 D post-challenge; the bacterial counts in spleen contents in TBA were lower (P < 0.05) than in CCA at 7 D post-challenge. Tumor necrosis factor-α contents in TBA and CCA were lower (P < 0.05) than those in PC. Also, the ratio of villus height to crypt depth in the ileum of CCA was higher (P < 0.05) than that of PC and ENR; however, there was no difference in the secretory IgA content of the jejunum among the groups. In conclusion, EOA had a bacteriostatic effect on S. Enteritidis, and the effect of the thymol-benzoic acid complex surpassed that of the cinnamaldehyde-caproic acid complex. Therefore, EOA may act as an effective antibiotic substitute for animals in the prevention and treatment of Salmonella.

[In vitro and in vivo effects of 5-aminolevulinic acid-mediated photodynamic therapy against oral squamous cell carcinoma].

Objective: To investigate the in vitro and in vivo effects of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy against oral squamous cell carcinoma (OSCC) and preliminarily explore the possible mechanisms. Methods: SCC25 cells were divided into the control group (5-ALA of 0 mg/L) and the experimental group (5-ALA of 10, 25, 50, 100 and 150 mg/L). The production of protoporphyrin Ⅸ (PpⅨ) induced by 5-ALA in SCC25 cells was detected using the flow cytometry. SCC25 cells were divided into the control group (5-ALA of 0 mg/L), lazer alone group, 5-ALA alone group (5-ALA of 100 mg/L) and the 5-ALA combined with laser irradiation group (5-ALA of 5, 10, 25, 50 and 100 mg/L), the cytotoxicity of 5-ALA combined with laser irradiation (wave length 635 nm, power density 87 mW/cm(2) and laser dose 10.4 J/cm(2)) was evaluated in SCC25 cells using the methyl thiazolyltetrazolium assay (incubation times of 4, 8 and 12 h in each group) and the induction effect of combination treatment on the cell apoptosis was assessed by the flow cytometry (incubation time of 12 h in each group). The intracellular production of reactive oxygen species (ROS) triggered by 5-ALA combined with laser irradiation was determined using a fluorescence probe method (incubation time of 12 h in each group). A mouse OSCC xenograft model bearing SCC25 tumor was built, and the mice were divided into control group (saline), 5-ALA group (5-ALA of 50 mg/kg) and 5-ALA combined with laser irradiation group (5-ALA of 10, 25 and 50 mg/kg). Antitumor effect of 5-ALA combined with laser irradiation (wave length 635 nm, power density 158 mW/cm(2) and laser dose 94.8 J/cm(2)) was further measured. Results: 5-ALA induced the production of PpⅨ in SCC25 cells in a drug concentration (0-150 mg/L)-and incubation time (0-24 h)-dependent manner. When the 5-ALA concentration was 100 mg/L, the intracellular PpⅨ production was in a relatively stable state. Cell viability and apoptosis rate of 5, 10, 25, 50, 100 mg/L 5-ALA combined with laser irradiation are, respectively, (82.3±5.2)%, (3.13±0.38)%; (74.6±9.3)%, (5.38±0.55)%; (38.3±9.7)%, (17.97±2.72)%; (9.2±3.8)%, (24.47±3.37)%; (7.2±0.8)%, (43.01±5.96)%, which indicated that 5-ALA combined with laser irradiation notably inhibited the growth of SCC25 cells and also induced significant cell apoptosis compared with the control group [(96.3±6.0)%, (0.35±0.13)%, P<0.05]. After combination treatment (5-ALA of 5, 10, 25, 50 and 100 mg/L combined with laser irradiation, the mean fluorescence intensity of dichlorofluorescein is (1.46±0.12)×10(4), (2.16±0.30)×10(4), (3.57±0.34)×10(4), (81.70±13.05)×10(4), (113.00±7.35)×10(4), respectively, a large amount of ROS was produced in SCC25 cells compared with the control group [(0.96±0.15) ×10(4), P<0.05], which was in positive correlation with the intracellular PpⅨ content. 5-ALA (concentration of 10, 25 and 50 mg/kg) combined with laser irradiation greatly suppressed the tumor growth in SCC25 tumor-bearing mice compared to the control group (P<0.05). Conclusions: 5-ALA-mediated photodynamic therapy can trigger the generation of intracellular ROS that has significant cytotoxicity and apoptosis induction effect, and thus inhibit the tumor growth both in vitro and in vivo.

A comparison of methods to determine amino acid availability of feedstuffs in cecectomized ganders.

Experiments were conducted to compare endogenous amino acid losses and the true amino acid availability (TAAA) of 3 feedstuffs by using methods involving a short-term fasting and an N-free diet with cecectomized ganders. Diets were formulated to contain soybean meal, fish meal, and cottonseed meal as the sole source of protein. A precision-fed assay was used in which each feed sample was precise-fed (60 g) to geese and excreta were collected for 48 h. A N-free diet and fasting methods were used to evaluate the endogenous amino acid losses. Endogenous losses of 3 amino acids were significantly different (P < 0.01) with the N-free diet and fasting methods. The TAAA of soybean meal, fish meal, and cottonseed meal determined by N-free diet method ranged from 84.49 to 97.09%, 89.18 to 98.16%, and 77.09 to 98.32%, respectively. The TAAA of these 3 diets determined by the fasting method ranged from 83.50 to 97.77%, 88.08 to 99.60%, and 76.09 to 98.09%, respectively. However, there were only a few small differences (P > 0.05) between methods in each amino acid. In conclusion, there was no difference in determination of the amino acid availability of these feedstuffs using cecectomized ganders between the N-free diet and fasting methods.

The influence of caecectomy on amino acid availability of three feedstuffs for ganders.

1. Experiments were conducted to assess the influence of caecectomy on amino acid availability (AAA) of three feedstuffs for goose. 2. Nine caecectomised and 9 intact Yangzhou ganders, 24 weeks old, were used in these experiments. Fish meal, soybean meal and cottonseed meal were used as the sole source of protein. The endogenous amino acid (AA) losses were evaluated by a nitrogen (N)-free diet method. The influence of caecectomy on apparent amino acid availability (AAAA) in fish meal, soybean meal and cottonseed meal was assessed in experiment 1 and true amino acid availability (TAAA) of three protein diets was determined in experiment 2. 3. Results showed that, in the soybean meal and cottonseed meal, the AAAA and TAAA of most AA determined by the intact ganders were higher than in the caecectomised ganders; in the fish meal, the AAAA and TAAA of most AA determined by the intact ganders were lower than in the caecectomised ganders. 4. Results of the present study suggest that the effect of caecectomy on AAA in geese was dependent on the feedstuff assayed, and it was better to use caecectomised poultry for AAA assessment.

Elevated and absent pRb expression is associated with bladder cancer progression and has cooperative effects with p53.

Rb protein (pRb) expression was evaluated in 185 cases of transitional cell carcinoma of the bladder from patients that underwent radical cystectomy. Tumors were stratified into three categories based on the percentage of nuclei expressing pRb: (a) 0, 0% of tumor cells showing nuclear reactivity; (b) 1+, 1-50% of tumor cells showing nuclear reactivity; and (c) 2+, >50% of tumor cells showing nuclear reactivity. Cases with undetectable (pRb 0) and high (pRb 2+) pRb reactivity had identical rates of recurrence. These cases had significantly higher recurrence (P = 0.0001) and lower survival rates (P = 0.0002) compared to cases with moderate (pRb 1+) pRb reactivity, indicating that high levels of pRb expression may reflect a dysfunctional (altered) Rb pathway. The tumors were also examined for alterations in p53 expression; patients with tumors altered in both p53 and pRb had significantly increased rates of recurrence (P < 0.0001) and survival (P < 0.0001) compared to patients with no alterations in either p53 or pRb; patients with alterations in only one of these proteins had intermediate rates of recurrence and survival. These results suggest that: (a) bladder cancers with high pRb expression do not show the tumor suppressor effects of the protein; and (b) alteration in both p53 and pRb may act in cooperative or synergistic ways to promote tumor progression.

Nitric oxide, prostanoids, cyclooxygenase, and angiogenesis in colon and breast cancer.

PURPOSE: Several studies have shown an overexpression of cyclooxygenase-2 (COX-2) and elevated levels of prostacyclin (PGI(2)) and thromboxane (TXA(2)) in colon cancer. In this report, we determined the distribution of inducible form of nitric oxide synthase (iNOS), PGI(2), and TXA(2) in cancerous and adjoining areas of specimens from human colon and breast cancer obtained during surgery. Additionally, we investigated differences in expression and histological localization of COX-2 in colon and breast cancer. EXPERIMENTAL DESIGN: Specimens were obtained during surgery, one centrally located, the second from an adjacent, cancer-free area. Activity of iNOS was determined, using the conversion of L-[(14)C]arginine to L-[(14)C]citrulline. PGI(2) and TXA(2) were measured as their stable metabolites, using enzyme immunoassay. A standard immunoperoxidase method was used for immunohistochemical expression of COX-2. RESULTS: Significant differences in iNOS, PGI(2), and TXA(2) expressions between colon and breast cancer were noted, with an enhanced expression of COX-2 in colon cancer, including the cancerous, adjoining, and stromatous fields. CONCLUSIONS: Increased expression of iNOS and production of prostanoids in colon cancer parallels the increase in COX-2, confirming the importance of this enzyme in colon cancer. The overexpression of COX-2, prostanoids, and nitric oxide in areas adjoining the tumor indicates increased metastatic potential for neoplastic cells in this area. Inflammatory changes in the tissue adjoining the cancer may play a role. COX-2 may result in the formation of new blood vessels and the spread of cancer.

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

Antigen retrieval techniques: current perspectives.

Development of the antigen retrieval (AR) technique, a simple method of boiling archival paraffin-embedded tissue sections in water to enhance the signal of immunohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide application of IHC in research and clinical pathology. On the basis of thousands of articles and many reviews, a book has recently been published that summarizes basic principles for practice and further development of the AR technique. Major topics with respect to several critical issues, such as the definition, application, technical principles, and further studies of the AR technique, are highlighted in this article. In particular, a further application of the heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to proteins, and standardization of routine IHC based on the AR technique in terms of a test battery approach, are also addressed. Furthermore, understanding the mechanism of the AR technique may shed light on facilitating the development of molecular morphology.

Antigen retrieval immunohistochemistry: past, present, and future.

The antigen retrieval (AR) technique, which is predominantly based on high-temperature heating of tissues, is used as a non-enzymatic pretreatment for immunohistochemical staining of formalin-fixed, paraffin-embedded tissue sections. It has been widely applied in pathology and analytical morphology. The existence of a growing body of literature on the AR technique raises a number of interesting issues for the further development of AR. These issues include the use of a "test battery" and the concept of "maximal retrieval" applied to the selection of optimal test protocols for the standardization of AR.

Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies.

Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.