Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance. METHODS: We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab. RESULTS: Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting. CONCLUSIONS: Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.

Tumour-infiltrating CD8+ lymphocytes as an independent predictive factor for pathological complete response to primary systemic therapy in breast cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are known to be associated with response to primary systemic therapy (PST) in breast cancer. This study was conducted to assess the association of TIL subsets with pathological complete response (pCR) after PST in breast cancer in relation to breast cancer subtype, breast cancer stem cell (BCSC) phenotype and epithelial-mesenchymal transition (EMT). METHODS: The pre-chemotherapeutic biopsy specimens of 153 breast cancer patients who underwent surgical resection after anthracycline- or anthracycline/taxane-based PST were analysed. TIL subsets (CD4+, CD8+, and FOXP3+ TILs), BCSC phenotype, and the expression of EMT markers were evaluated by immunohistochemistry and were correlated with pCR after PST. RESULTS: Infiltration of CD4+ and CD8+ T lymphocytes was closely correlated with BCSC phenotype and EMT. High levels of CD4+, CD8+, and FOXP3+ TILs were associated with pCR, and CD8+ TILs were found to be an independent predictive factor for pCR. In addition, CD8+ TILs were associated with pCR irrespective of breast cancer subtype, CD44+/CD24- phenotype, EMT, and chemotherapeutic regimen in subgroup analyses. CONCLUSION: These findings indicate that CD8+ cytotoxic T lymphocytes are a key component of TILs associated with chemo-response and can be used as a reliable predictor of response to anthracycline- or anthracycline/taxane-based PST in breast cancer.

Staphylococcus aureus-derived extracellular vesicles induce neutrophilic pulmonary inflammation via both Th1 and Th17 cell responses.

BACKGROUND: Recent evidence indicates that Staphylococcus aureus, one of the most important human pathogens, secretes vesicles into the extracellular milieu. OBJECTIVE: To evaluate whether inhalation of S. aureus-derived extracellular vesicles (EV) is causally related to the pathogenesis of inflammatory pulmonary diseases. METHODS: Staphylococcus aureus EV were prepared by sequential ultrafiltration and ultracentrifugation. The innate immune response was evaluated in vitro after the application of EV to airway epithelial cells and alveolar macrophages. In vivo innate and adaptive immune responses were evaluated after airway exposure to EV. Adjuvant effects of EV on the development of hypersensitivity to inhaled allergens were also evaluated after airway sensitization with S. aureus EV and ovalbumin (OVA). RESULTS: Staphylococcus aureus and S. aureus EV were detected in house dust. Alveolar macrophages produced both tumor necrosis α (TNF-α) and interleukin 6 (IL-6) after in vitro stimulation with S. aureus EV, whereas airway epithelial cells produced only IL-6. Repeated airway exposure to S. aureus EV induced both Th1 and Th17 cell responses and neutrophilic pulmonary inflammation, mainly via a Toll-like receptor 2 (TLR2)-dependent mechanism. In terms of adjuvant effects, airway sensitization with S. aureus EV and OVA resulted in neutrophilic pulmonary inflammation after OVA challenge alone. This phenotype was partly reversed by the absence of interferon γ (IFN-γ) or IL-17. CONCLUSION: Staphylococcus aureus EV can induce Th1 and Th17 neutrophilic pulmonary inflammation, mainly in a TLR2-dependent manner. Additionally, S. aureus EV enhance the development of airway hypersensitivity to inhaled allergens.

Diagnosis of Critical Illness Myopathy After Liver Transplantation and Muscle Condition Monitoring: A Case Report.

BACKGROUND: Intensive care unit-acquired weakness (ICUAW) can occur after liver transplantation. Early diagnosis of ICUAW and monitoring of muscle condition during rehabilitation are helpful in improving functional recovery. METHODS AND MATERIALS: A 47-year-old man with liver cirrhosis developed limb weakness after liver transplantation. The patient had a Medical Research Council sum score of 2 weeks post-liver transplantation with marked proximal limb weakness. Direct muscle stimulation was performed on the right tibialis anterior muscle; the nerve-to-muscle ratio of compound muscle action potentials was 0.96, which indicated critical illness myopathy. Fatigue analysis using surface electromyography was performed 4 times after liver transplantation. RESULTS AND CONCLUSIONS: The maximal voluntary contraction tended to increase during rehabilitation, whereas the percentage of maximal voluntary contraction tended to decrease, indicating that muscle strength was increased. The fatigue index gradually decreased, showing that muscle endurance had improved along with strength. Muscle fatigue can be evaluated during rehabilitation using surface electromyography to prevent damage of the impaired muscle and to control exercise intensity. Early diagnosis of ICUAW and evaluation of muscle fatigue during rehabilitation will ensure a better prognosis for patients with ICUAW.

Adenosine A2A receptor and ERK-driven impulsivity potentiates hippocampal neuroblast proliferation.

Dampened adenosine A2A receptor (A2AR) function has been implicated in addiction through enhancement of goal-directed behaviors. However, the contribution of the A2AR to the control of impulsive reward seeking remains unknown. Using mice that were exposed to differential reward of low rate (DRL) schedules during Pavlovian-conditioning, second-order schedule discrimination, and the 5-choice serial reaction time task (5-CSRTT), we demonstrate that deficits of A2AR function promote impulsive responses. Antagonism of the A2AR lowered ERK1 and ERK2 phosphorylation in the dorsal hippocampus (dHip) and potentiated impulsivity during Pavlovian-conditioning and the 5-CSRTT. Remarkably, inhibition of ERK1 and ERK2 phosphorylation by U0126 in the dHip prior to Pavlovian-conditioning exacerbated impulsive reward seeking. Moreover, we found decreased A2AR expression, and reduced ERK1 and ERK2 phosphorylation in the dHip of equilibrative nucleoside transporter type 1 (ENT1-/-) null mice, which displayed exacerbated impulsivity. To determine whether impulsive response behavior is associated with hippocampal neuroblast development, we investigated expression of BrdU+ and doublecortin (DCX+) following 5-CSRTT testing. These studies revealed that impulsive behavior driven by inhibition of the A2AR is accompanied by increased neuroblast proliferation in the hippocampus.

Treadmill running promotes functional recovery and decreases brain-derived neurotrophic factor mRNA expression following sciatic crushed nerve injury in rats.

AIM: Peripheral nerve injuries are commonly encountered clinical problems and often result in severe functional deficits. In the present study, the effects of treadmill running on the recovery rate of locomotor function and the expression of brain-derived neurotrophic factor (BDNF) mRNA following sciatic crushed nerve injury in rats were investigated. EXPERIMENTAL DESIGN: Comparative investigation was performed over 14 days. SETTING: Experimental animal laboratory. PARTICIPANTS: 24 male Sprague-Dawley rats weighing 200+/-10 g. Animals were randomly assigned into 3 groups: the sham-operation group, the sciatic nerve injury group, and the sciatic nerve injury and running group. INTERVENTIONS: The right sciatic nerve was crushed for 30 s using a surgical clip. Rats of the running group were made to run on a treadmill for 30 min once a day for 12 consecutive days. MEASURES: Functional recovery was analyzed using a walking track analysis which can be quantified with the sciatic function index (SFI) and BDNF mRNA expression was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Sciatic crushed nerve injury showed characteristic gait changes showing decrease of SFI value and treadmill running significantly enhanced the SFI value. The level of BDNF mRNA expression was increased following sciatic crushed nerve injury and treadmill running significantly suppressed the sciatic nerve injury-induced increment of BDNF mRNA expression. CONCLUSIONS: It can be suggested that treadmill running after peripheral nerve injury is effective in the functional recovery by inhibition on the over-expression of BDNF mRNA.

IL-1ß in eosinophil-mediated small intestinal homeostasis and IgA production.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1ß (IL-1ß), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-ß, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-ß (transforming growth factor ß). GI eosinophils expressed a relatively high level of IL-1ß, and IL-1ß-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1ß in the small intestine.

Effects of alendronate on osteopenic postmenopausal Chinese women.

To evaluate the effects of alendronate on postmenopausal Chinese women with osteopenia, we treated 46 subjects daily with either 10 mg alendronate (N = 24) or placebo plus 500 mg calcium supplement (N = 22), and measured their bone mineral density (BMD) at the lumbar spine and hip, and urinary bone resorption markers before, during, and after the 1 year treatment period. The bone markers included N-telopeptide of type I collagen (NTx) and deoxypyridinoline (Dpd); both were corrected by the concentration of creatinine in the same sample (NTx/Cr and Dpd/Cr). Both NTx/Cr and Dpd/Cr decreased significantly by 44% and 28%, respectively (p < 0.05 for both), in 1 month in the active treatment group but did not change in the placebo group. BMD at the spine, femoral neck, trochanter, and Ward's triangle increased significantly by 6 months and showed a further increase through month 12 at the spine in the alendronate-treated group. Relative to the placebo group, BMD changes at various sites in the alendronate-treated group were higher at 12 months by 6%-11%. Thus, our data suggest that 10 mg alendronate daily resulted in significant increases in spine and hip BMD, and decreases of urinary resorption markers in the osteopenic postmenopausal Chinese women studied. The amplitude of responses was higher than in previous reports in the USA and Europe.

Quantitative analysis of polyvinyl alcohol on the surface of poly(D, L-lactide-co-glycolide) microparticles prepared by solvent evaporation method: effect of particle size and PVA concentration.

Polyvinyl alcohol (PVA) is an emulsion stabilizer that is used in the solvent evaporation method for poly(D,L-lactide-co-glycolide) (PLG) microparticles preparation. In this study, the surface binding of PVA on PLG microparticles was quantitatively examined by employing gel permeation chromatography. The PVA binding can affect hydrophobicity and digestibility of the microparticle surface. GPC analysis detected the presence of PVA bound on the microparticle surfaces in an irreversible manner. The PVA content per weight of microparticle increased with the specific surface area as the particle size decreased. The surface PVA density, which is the PVA content per unit surface area of microparticle, changed with particle size in a biphasic manner. In the size range larger than 1 microm, the surface PVA density increased from 0.8 mg/m2 to 2.2 mg/m2 as the particle size decreased. But below 1 microm, the surface PVA density remained unchanged at about 1.8 mg/m2. The surface PVA density of PLG microparticles prepared in different PVA concentrations (0.1, 1, 5, 10% w/v) showed similar values and size dependency, indicating that the PVA concentration of the continuous phase did not affect the surface PVA density of PLG microparticles. These data suggest that the influence of the surface PVA layer would be larger in the smaller particles, but be independent of the PVA concentration of the continuous phase in the manufacturing process.

Auricular acupuncture decreases neuropeptide Y expression in the hypothalamus of food-deprived Sprague-Dawley rats.

The aim of the present study was to investigate whether stimulation of auricular acupuncture point has any effects on the expression of neuropeptide Y (NPY), appetite-inducing factor particularly abundant in the mammalian hypothalamus. In food-deprived condition, enhanced NPY expression was detected in both the arcuate nucleus (ARN) and the paraventricular nucleus (PVN) of the hypothalamus via immunohistochemistry in Sprague-Dawley rats. Needling the unfed rats on the auricular point resulted in decreased NPY levels in both the ARN and PVN, while it increased NPY levels in the ARN and PVN of fed rats. The present findings indicate that auricular acupuncture may affect NPY expression in the ARN and PVN of the hypothalamus.

Caffeine inhibits exercise-induced increase in tryptophan hydroxylase expression in dorsal and median raphe of Sprague-Dawley rats.

Effect of caffeine on the expression of tryptophan hydroxylase (TPH), rate limiting enzyme of serotonin synthesis, in dorsal and median raphe was investigated via immunohistochemistry. In exercise groups, Sprague-Dawley rats were put on treadmill running for 30 min per day for 6 consecutive days. On the seventh day, animals of control-with-caffeine group were injected subcutaneously with 4 mg/kg caffeine, while control-without-caffeine group were injected with 0.9% NaCl, sacrificed 2 h later. Exercise-with-caffeine group and exercise-without-caffeine group were injected with caffeine and NaCl, respectively; all-out time was determined 1 h after injection, and then sacrificed. Caffeine increased all-out time in exercised rats, and inhibited the exercise-induced elevation in TPH expression. The suppressive effect of caffeine on TPH expression in exercised rats can be suggested as one possible ergogenic mechanism of caffeine.

Treadmill exercise increases cell proliferation in hippocampal dentate gyrus in alcohol-intoxicated rats.

AIM: Effect of treadmill exercise on hippocampal cell proliferation under normal conditions has been well documented; however, this effect under alcohol intoxication conditions is not clarified, yet. In the present study, the effect of treadmill exercise on cell proliferation in the dentate gyrus in alcohol-intoxicated rats was investigated. EXPERIMENTAL DESIGN: comparative investigation on number of 5-bromo-2'-deoxyuridine (BrdU)-positive cells in the dentate gyrus 8 days after commencement. SETTING: animal laboratory. PARTICIPANTS: male Sprague-Dawley rats of 5 weeks in age weighing 150+/-10 g. INTERVENTION: animals were divided into 4 groups: the control-rest group, the control-exercise group, the alcohol-treated-rest group, and the alcohol-treated-exercise group. Animals of the alcohol-treated groups were injected intraperitoneally with alcohol (2 g/kg) once a day for 3 days. All animals were injected BrdU (50 mg/kg) intraperitoneally, and rats of exercise groups were made to run on treadmill for 30 min each day for 5 days following alcohol administration. MEASURES: mean number of BrdU-positive cells in dentate gyrus was observed via immunohistochemistry. RESULTS: Treadmill exercise significantly increased the number of BrdU-positive cells in the dentate gyrus. Also, treatment with alcohol for 3 days inhibited cell proliferation and treadmill exercise alleviated alcohol-induced inhibition of new cell formation. CONCLUSION: These results suggest the possibility that treadmill exercise may help in improvement following alcohol-induced brain damage.

Comparison of peritoneal dialysis and hemodialysis after kidney transplant failure.

BACKGROUND: Patients with a failed kidney transplant represent a unique chronic kidney disease population that is increasing in number and is at high risk of morbidity and mortality. Among transplant-naïve patients, those treated with peritoneal dialysis (PD) show an early survival advantage compared with those treated with hemodialysis (HD). But any advantage of PD after allograft failure is unknown. The aim of this study was to investigate the clinical outcomes of patients with failed allografts according to the type of dialysis modality. METHOD: We reviewed medical records of patients who initiated dialysis after kidney transplant failure from November 1982 to May 2011. Demographics features, clinical data, and survival outcomes were compared between PD and HD patients who had experienced allograft failure. RESULTS: The 182 patients with failed allografts showed the most common cause to be chronic rejection. The median duration of function before allograft failure was 74.0 months. After allograft failure, 145 (79.7%) patients returned to HD and 37 (20.3%) to PD. Twenty-three patients (12.6%) died over the median 69.1 months duration of follow-up. During the observation period, 16 HD (11%) and 7 PD (8.9%) patients died. The survival rates of PD patients at 1 year were 91.2% and 84.4%, respectively, at 1 and 3 years, and those of HD patients 94.8% and 88.9%. There was no significant difference in the survivals of the 2 groups. CONCLUSIONS: The study suggests that the outcome of patients starting PD after kidney transplant failure was similar to those starting HD. Therefore, PD can be regarded to be a good treatment option for patients returning to dialysis after kidney transplant failure.

Identification of a pivotal endocytosis motif in c-Met and selective modulation of HGF-dependent aggressiveness of cancer using the 16-mer endocytic peptide.

Since c-Met has an important role in the development of cancer, it is considered as an attractive target for cancer therapy. Although molecular mechanisms for oncogenic property of c-Met have been actively investigated, regulatory elements for c-Met endocytosis and its effect on c-Met signaling remain unclear. In this study, we identified a pivotal endocytic motif in c-Met and tested it for selective modulation of HGF-induced c-Met response. Using various chimeric constructs with the cytoplasmic tail of c-Met, we were able to demonstrate that a dileucine motif located in the C-terminus of c-Met acts to regulate its endocytosis. Synthetic peptide Ant-3S, consisting of antennapedia-derived protein transduction domain (designated as Ant) and c-Met-derived 16 amino-acids (designated as 3S, spanning amino-acids 1378 to 1393), rapidly moved into cancer cells and disrupted c-Met trafficking. Importantly, an extension of c-Met retention time on the membrane by Ant-3S peptide significantly decreased phosphorylation-dependent c-Met signal transduction. Additionally, the peptide effectively inhibited HGF-induced cell growth, scattering and migration. The underlying molecular mechanism for these observations has been investigated and revealed that the dileucine motif interacts with endocytic machinery, including adaptin ß and caveolin-1, for sustained and enhanced signal transduction. Finally, Ant-3S peptide specifically blocked internalization of interleukin-2 receptor α-subunit/3S chimeric protein, but not the other receptors, including Glut4, Glut8 and transferrin receptor. Such results indicate the presence of a selective endocytic assembly for c-Met. It also suggests a potential for c-Met-specific anti-cancer therapy using the identified endocytic motif in this study.

Localized CT-guided irradiation inhibits neurogenesis in specific regions of the adult mouse brain.

Radiation is used in the study of neurogenesis in the adult mouse both as a model for patients undergoing radiation therapy for CNS malignancies and as a tool to interrupt neurogenesis. We describe the use of a dedicated CT-guided precision device to irradiate specific sub-regions of the adult mouse brain. Improved CT visualization was accomplished with intrathecal injection of iodinated contrast agent, which enhances the lateral ventricles. T2-weighted MRI images were also used for target localization. Visualization of delivered beams (10 Gy) in tissue was accomplished with immunohistochemical staining for the protein γ-H2AX, a marker of DNA double-strand breaks. γ-H2AX stains showed that the lateral ventricle wall could be targeted with an accuracy of 0.19 mm (n = 10). In the hippocampus, γ-H2AX staining showed that the dentate gyrus can be irradiated unilaterally with a localized arc treatment. This resulted in a significant decrease of proliferative neural progenitor cells as measured by Ki-67 staining (P < 0.001) while leaving the contralateral side intact. Two months after localized irradiation, neurogenesis was significantly inhibited in the irradiated region as seen with EdU/NeuN double labeling (P < 0.001). Localized radiation in the rodent brain is a promising new tool for the study of neurogenesis.

Radiolytic studies of trimethylamine dehydrogenase. Spectral deconvolution of the neutral and anionic flavin semiquinone, and determination of rate constants for electron transfer in the one-electron reduced enzyme.

Trimethylamine dehydrogenase from the pseudomonad Methylophilus methylotrophus has been examined using the technique of pulse radiolysis to rapidly introduce a single reducing equivalent into the enzyme. Using enzyme that has had its iron-sulfur center rendered redox-inert by prior reaction with ferricenium hexafluorophosphate, we determined the spectral change associated with formation of both the anionic and neutral forms that were generated at high and low pH, respectively, of the unique 6-cysteinyl-FMN of the enzyme. With native enzyme, electron transfer was observed within the radiolytically generated one-electron reduced enzyme but only at low pH (6.0). The kinetics and thermodynamics of this electron transfer in one-electron reduced enzyme may be compared with that studied previously in the two-electron reduced enzyme. In contrast to previous studies with two-electron reduced enzyme in which a pK(a) of approximately 8 was determined for the flavin semiquinone, in the one-electron reduced enzyme the semiquinone was not substantially protonated even at pH 6. 0. These results indicate that reduction of the iron-sulfur center of the enzyme significantly decreases the pK(a) of the flavin semiquinone of the active site. This provides further evidence, in conjunction with the strong magnetic interaction known to exist between the centers in the two-electron reduced enzyme, that the two redox-active centers in trimethylamine dehydrogenase are in intimate contact with one another in the active site of the enzyme.

Formation of W(3)A(1) electron-transferring flavoprotein (ETF) hydroquinone in the trimethylamine dehydrogenase x ETF protein complex.

The electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (TMADH), or reaction with strong reducing reagents such as sodium dithionite). In the present study, we demonstrate that ETF can be reduced fully to its hydroquinone form both enzymatically and chemically when it is in complex with TMADH. Quantitative titration of the TMADH x ETF protein complex with sodium dithionite shows that a total of five electrons are taken up by the system, indicating that full reduction of ETF occurs within the complex. The results indicate that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, a conclusion further supported by the observation of a spectral change upon formation of the TMADH x ETF complex that is due to a change in the environment of the FAD of ETF. The results are discussed in the context of ETF undergoing a conformational change during formation of the TMADH x ETF electron transfer complex, which modulates the spectral and oxidation-reduction properties of ETF such that full reduction of the protein can take place.

Microcystin production by Microcystis aeruginosa in a phosphorus-limited chemostat.

The production of microcystins (MC) from Microcystis aeruginosa UTEX 2388 was investigated in a P-limited continuous culture. MC (MC-LR, MC-RR, and MC-YR) from lyophilized M. aeruginosa were extracted with 5% acetic acid, purified by a Sep-Pak C(18) cartridge, and then analyzed by high-performance liquid chromatography with a UV detector and Nucleosil C(18) reverse-phase column. The specific growth rate (mu) of M. aeruginosa was within the range of 0.1 to 0.8/day and was a function of the cellular P content under a P limitation. The N/P atomic ratio of steady-state cells in a P-limited medium varied from 24 to 15 with an increasing mu. The MC-LR and MC-RR contents on a dry weight basis were highest at mu of 0.1/day at 339 and 774 microg g(-1), respectively, while MC-YR was not detected. The MC content of M. aeruginosa was higher at a lower mu, whereas the MC-producing rate was linearly proportional to mu. The C fixation rate at an ambient irradiance (160 microeinsteins m(-2) s(-1)) increased with mu. The ratios of the MC-producing rate to the C fixation rate were higher at a lower mu. Accordingly, the growth of M. aeruginosa was reduced under a P limitation due to a low C fixation rate, whereas the MC content was higher. Consequently, increases in the MC content per dry weight along with the production of the more toxic form, MC-LR, were observed under more P-limited conditions.

Development of antigen induced colitis in SCID mice reconstituted with spleen derived memory type CD4(+) CD45RB(+) T cells.

BACKGROUND AND AIMS: Enteric bacterial and/or food antigens may be crucial in the development of colitis but little is known of the exact mechanism of antigen specific reactions in this condition. The aim of this study was to determine whether systemically primed antigen specific CD4(+) T cells containing both CD45RB(high) and CD45RB(low) populations participate as a pathogenic subset that in turn leads to inflammatory reactions selectively in the large intestine. METHODS: SCID mice were reconstituted with splenic CD4(+) CD45RB(+) T cells or CD4(+) CD45RB(low) T cells isolated from donor mice systemically primed with ovalbumin (OVA) plus CFA. The reconstituted mice were then fed OVA for several weeks. RESULTS: Reconstitution of SCID mice with OVA primed splenic CD4(+) T cells, containing populations of CD45RB(high) and CD45RB(low), resulted in the development of colitis by 4-5 weeks following repeated administration of oral OVA. Histopathological study revealed thickened wall, inflammatory cell infiltration, crypt elongation, and loss of goblet cells in the large intestine. The CD4(+) CD45RB(low) population of cells extracted from the affected large intestine secreted high levels of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) at the protein and mRNA levels. Administration of neutralising antibodies to TNF-alpha, but not to IFN-gamma, prevented the development of colitis. Furthermore, adoptive transfer with OVA primed splenic CD4(+) CD45RB(low) T cells evoked severe colitis. CONCLUSIONS: These results demonstrate that systemically primed activated/memory CD4(+) CD45RB(low) T cells can mediate the development of specific antigen induced colitis in SCID mice, and also that TNF-alpha is critical in the induction of this type of colitis. Our results contrast with those from studies in some colitis models in which CD45RB(low) T cells appeared to prevent colitis through secretion of immunosuppressive cytokines.