BACKGROUNDRecent studies have shown that T helper type-2 (Th2) cells can induce the apoptosis of CD4+CD25+ Treg cells or resist the immunosuppressive effect of Treg cells. We hypothesize that an imbalance of Th2/Treg is present in patients with allergic asthma.

METHODSTwenty-two patients with mild asthma, 17 patients with moderate to severe asthma, and 20 healthy donors were enrolled. All patients were allergic to house dust mites. The proportion of peripheral blood CD4+CD25+ Treg cells and Th2 cells were determined by flow cytometry. The concentration of interleukin (IL)-10, transforming growth factor (TGF)-β and IL-4 in plasma was determined by enzyme linked immunosorbent assay. In these subjects, peripheral blood mononuclear cells from 17 mild asthmatic patients, 13 moderate to severe asthmatic patients and 14 healthy donors were acquired and expression of forkhead box P3 (Foxp3) and GATA-3 mRNA was detected by reverse-transcriptase polymerase chain reaction.

RESULTSCompared with healthy donors and patients with mild asthma, the percent of CD4+CD25+ Treg cells and plasma IL-10 levels were decreased in patients with moderate to severe asthma. There were no significant differences in Foxp3 mRNA expression among three groups, but a downward trend seen among patients with asthma. However, the percent of Th2 cells, IL-4 levels and expression of GATA-3 mRNA was markedly higher in patients with mild and moderate to severe asthma than in the control group. The ratio of Th2/Treg and their cytokines was increased in allergic asthma, especially for moderate to severe asthma. The ratio of GATA-3/Foxp3 mRNA was also increased in allergic asthma. In patients with moderate to severe asthma, the percentage of peripheral blood Treg cells was negatively correlated to the percentage of Th2 cells and IL-4 levels.

CONCLUSIONSThe decline of CD4+CD25+ Treg cells in patients with moderate to severe asthma may play an important role in progress of the disease. Furthermore, the deficiency of CD4+CD25+ Treg cells was associated with the over-expression of Th2 response.

Asthma ; etiology ; immunology ; Cytokines ; blood ; Forkhead Transcription Factors ; genetics ; GATA3 Transcription Factor ; genetics ; Humans ; RNA, Messenger ; analysis ; T-Lymphocytes, Regulatory ; immunology ; Th2 Cells ; immunology

OBJECTIVEWe have studied 4 generations 12 patients in a family which has blepharophimosis-ptosis-epicanthus-inversus syndrome (BPES) for the gene, FOXL2, the group also have 12 normal members in this family and other 80 normal individuals for contrast.

METHODSThe FOXL2 gene was amplified by polymerase chain reaction and then analyzed by direct genomic sequencing.

RESULTSA 892C > T at nucleotides in FOXL2 was found in the twelve affected patients. No mutations was found in any of the health members in the family.

CONCLUSIONSFOXL2 may be a important pathogenesis for the disease in this Chinese family.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Blepharophimosis ; ethnology ; genetics ; Child ; Child, Preschool ; Female ; Forkhead Box Protein L2 ; Forkhead Transcription Factors ; genetics ; Genotype ; Humans ; Male ; Middle Aged ; Mutation ; Pedigree ; Phenotype ; Sequence Analysis, DNA ; Syndrome ; Young Adult

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.
Aged ; Disease-Free Survival ; Female ; Forkhead Transcription Factors/*analysis/genetics ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Nerve Tissue Proteins/*analysis/genetics ; Odds Ratio ; Prognosis ; RNA, Messenger/metabolism ; ROC Curve ; Tumor Markers, Biological/*analysis ; Urinary Bladder Neoplasms/metabolism/*pathology

Forkhead box O-class 1 (FOXO1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. Its functions are modulated by forkhead box G1 (FOXG1), which acts as a transcriptional repressor with oncogenic potential. Real-time PCR and immunohistochemical staining were performed in 174 primary bladder cancer specimens and 21 normal bladder mucosae to evaluate these genes. FOXO1 and FOXG1 mRNA expression in cancer tissues were higher than in normal mucosae (each P<0.001). FOXO1 mRNA levels were significantly higher in samples of non-progressed patients (P<0.001), but FOXG1 were enhanced in those of progressed patients (P=0.019). On univariate analysis, FOXO1 mRNA expression was significantly associated with grade, stage, recurrence, progression and survival (each P<0.05). On multivariate analysis, increased FOXO1 mRNA expression was associated with both reduced disease progression (odds ratio [OR], 0.367; 95% confidence interval [CI], 0.163-0.826, P=0.015) and enhanced disease-free survival (OR, 3.262; 95% CI, 1.361-7.820, P=0.008). At a median follow-up of 33 months (range 2 to 156), the patients with a high FOXO1 mRNA expression had a significantly prolonged survival (P=0.001). Immunohistochemical findings of FOXO1 were generally concordant with mRNA expression levels. In conclusion, FOXO1 may be a promising marker for predicting progression in human bladder cancers.
Aged ; Disease-Free Survival ; Female ; Forkhead Transcription Factors/*analysis/genetics ; Humans ; Male ; Middle Aged ; Neoplasm Staging ; Nerve Tissue Proteins/*analysis/genetics ; Odds Ratio ; Prognosis ; RNA, Messenger/metabolism ; ROC Curve ; Tumor Markers, Biological/*analysis ; Urinary Bladder Neoplasms/metabolism/*pathology

OBJECTIVETo investigate variation of FOXP3 and it's expression in male children presented with severe and early-onset enteropathy, rash with or without insulin-dependent diabetes mellitus (IDDM).

METHODSFour male children presented with severe and early-onset enteropathy, rash, with or without IDDM were subjected to detection of FOXP3 expression on the PBMC by flow cytometry and FOXP3 gene analysis. The maternal gene analysis was subsequently performed once the variant FOXP3 gene was found. All 11 exons and splice sites within FOXP3 gene were amplified by polymerase chain reaction (PCR) from genomic DNA. Reverse transcription polymerase chain reaction was used to amplify the FOXP3 transcripts. Sequence analysis was performed directly on the bulk PCR products forwardly and reversely. The candidate mutation site was compared with that of 100 healthy controls to exclude polymorphism. Flow cytometry was used to determine FOXP3 expression on CD4+CD25+ T cells and the frequency of Tregs in CD4+ T cells.

RESULTSOne of the 4 patients showed a G13128A genetic variation in exon 11, which resulted in a Met370Ile substitution. No sequence variations were found at the same site in any of 100 healthy controls, indicating that the Met370Ile substitution is not a polymorphism but a novel missense mutation. The patient's mother was identified as a carrier for this mutation. There was no reduced frequency of Tregs in the peripheral blood of the patient and FOXP3 protein expression is normal as compared with controls.

CONCLUSIONA novel missense mutation of FOXP3 which causes IPEX phenotype was identified in a Chinese child according to immunologic screening and gene sequencing. Infants with early-onset IDDM and persistent diarrhea should be suspected as IPEX, FOXP3 gene analysis will be a reliable diagnostic approach to IPEX.

Child, Preschool ; DNA Mutational Analysis ; Diabetes Mellitus, Type 1 ; genetics ; immunology ; Forkhead Transcription Factors ; genetics ; Genes, X-Linked ; Genetic Diseases, X-Linked ; genetics ; immunology ; metabolism ; Humans ; Infant ; Intestinal Diseases ; genetics ; metabolism ; Male ; Mutation, Missense ; Syndrome ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo explore the role and mechanisms of FOXO3a nuclear translocation in neuronal apoptosis after hypoxia-ischemia (HI).

METHODSOne hundred and sixty 10-day-old Sprague-Dawly rats were randomly divided into two groups: HI and sham-operated. The right common carotid artery was ligated followed by hypoxia exposure for 2.5 hours in the HI group. The sham-operated group rats were not subjected to carotid artery ligation or hypoxia treatment. Rat cerebral cortex was collected at 0.5, 2, 4, 8 and 24 hours after hypoxia. Western blot was used to detect expression of total FOXO3a protein, pnuclear and cytoplasmic FOXO3a and Bim. TUNEL staining was used to detect apoptotic cells.

RESULTSThe nuclear protein of FOXO3a obviously increased from 0.5 to 24 hours after HI in a time-dependent manner compared with the sham-operated group (P<0.01). On the contrary, cytoplasmic protein evidently decreased from 0.5 to 24 hours in the HI group compared with the sham-operated group (P<0.01). Bim protein increased from 0.5 hour, peaked at 2 hours, started to decline at 4 hours (P<0.01), and returned to baseline level at 8 and 24 hours after HI in the HI group compared with the sham-operated group. TUNEL positive cells started to express at 4 hours, and peaked at 24 hours after HI (P<0.01). However, TUNEL positive cells were rarely found in the sham-operated group.

CONCLUSIONSHI induces FOXO3a translocation from cytoplasm to nucleus, and enhances protein expression of its target gene Bim in the neonatal rat brain. The upregulation of Bim expression might be related to neuronal apoptosis.

Animals ; Animals, Newborn ; Apoptosis ; Apoptosis Regulatory Proteins ; analysis ; Bcl-2-Like Protein 11 ; Female ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; physiology ; Hypoxia-Ischemia, Brain ; pathology ; Male ; Membrane Proteins ; analysis ; Neurons ; pathology ; Proto-Oncogene Proteins ; analysis ; Rats ; Rats, Sprague-Dawley

PURPOSE: To describe clinical findings in a Korean family with Axenfeld-Rieger syndrome. METHODS: A retrospective review of clinical data about patients with diagnosed Axenfeld-Rieger syndrome. Five affected members of the family underwent a complete ophthalmologic examination. We screened the forkhead box C1 gene and the pituitary homeobox 2 gene in patients. Peripheral blood leukocytes and buccal mucosal epithelial cells were obtained from seven members of a family with Axenfeld-Rieger syndrome. DNA was extracted and amplified by polymerase chain reaction, followed by direct sequencing. RESULTS: The affected members showed iris hypoplasia, iridocorneal adhesions, posterior embryotoxon, and advanced glaucoma in three generation. None had systemic anomalies. Two mutations including c.1362_1364insCGG and c.1142_1144insGGC were identified in forkhead box C1 in four affected family members. CONCLUSIONS: This study may help to understand clinical findings and prognosis for patients with Axenfeld-Rieger syndrome.
Aged, 80 and over ; Anterior Eye Segment/*abnormalities/metabolism ; DNA/*genetics ; DNA Mutational Analysis ; Eye Abnormalities/diagnosis/*genetics/metabolism ; Female ; Forkhead Transcription Factors/*genetics/metabolism ; Genetic Testing ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Male ; Middle Aged ; *Mutation ; Pedigree ; Retrospective Studies ; Transcription Factors/*genetics/metabolism ; Young Adult

OBJECTIVETo explore the impact of Foxp3 expression and CD(4)(+)CD(25)(+) regulatory T cells on pathogenesis of childhood asthma.

METHODSTotally 15 patients with acute asthma exacerbation, 15 children with asthma remission and 10 children who were hospitalized for skeleton deformity without atopic disorders or history of allergic diseases or respiratory infections within a month as controls were recruited in this study from Sep. 2004 to Mar. 2005. The percentage of CD(4)(+)CD(25)(+) T cells were detected by 2-color flow cytometry. The levels of interleukin (IL)-4, IL-10, interferon (IFN)-gamma, transforming growth factor (TGF)-beta in plasma and supernatant were assayed by ELISA. Both the asthmatic children and the control children were selected to induce sputum by hypertonic saline. Sputum was processed for detecting the expression of Foxp3-mRNA. The expression of Foxp3-mRNA in both sputum and PBMC was detected by semi-quantitative RT-PCR with beta-actin as internal control.

RESULTSThe percentage of CD(4)(+)CD(25)(+) regulatory T cells in exacerbation and remission asthmatic children was significantly lower than that of the control children both prestimulation [(10.1 +/- 2.1)% vs. (15.5 +/- 2.7)%, (11.7 +/- 2.5)% vs. (15.5 +/- 2.7)%, P < 0.05] and poststimulation with PHA [(12.4 +/- 2.3)% vs. (26.9 +/- 3.8)%, (17.3 +/- 3.2)% vs. (26.9 +/- 3.8)%, P < 0.05]. The percentage of CD(4)(+)CD(25)(+) regulatory T cells was significantly higher after PHA stimulation in normal children [(15.5 +/- 2.7)% vs. (26.9 +/- 3.8)%, P < 0.01]. The expression of Foxp3-mRNA (Foxp3/beta-actin) in asthmatic children was significantly lower than that in the control children in both PBMC and induced sputum. The expression of Foxp3-mRNA in PBMC was significantly higher after PHA stimulation in the control children (0.77 +/- 0.22 vs. 1.07 +/- 0.21, P < 0.05). However, there was no significant difference in Foxp3-mRNA expression in asthmatic children pre and post PHA stimulation. A significant positive correlation between the Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells was detected. The levels of IFN-gamma and TGF-beta were significantly lower in asthmatic children than those in the control children, and the levels of IFN-gamma and TGF-beta correlated positively with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells. The level of IL-4 both in plasma and supernatant was higher in asthmatic children. The levels of IL-10 was higher only in exacerbation than in control children, the levels of IL-4 and IL-10 had no correlation with Foxp3-mRNA expression and the percentage of CD(4)(+)CD(25)(+) regulatory T cells.

CONCLUSIONInsufficient secretion of TGF-beta, decreased Foxp3 expression, insufficient number of CD(4)(+)CD(25)(+) regulatory T cells and the defective ability of converting CD(4)(+)CD(25)(-) T cells to CD(4)(+)CD(25)(+) regulatory T cells might play an important role in pathogenesis of asthma.

Asthma ; physiopathology ; Case-Control Studies ; Child ; Cytokines ; analysis ; blood ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sputum ; chemistry ; T-Lymphocytes, Regulatory ; physiology

OBJECTIVETo investigate whether neuroblastoma cells LA-N-6 express Foxp3 and whether the expression of Foxp3 is sensitive to chemotherapy by cyclophosvnamide (CTX)and pirarubicin (THP).

METHODSExpression of Foxp3 on LA-N-6 cells was examined by flow cytometry analysis. The dose-effects of chemotherapy drugs including CTX and THP on LA-N-6 cells were investigated by MTT assay. The effects of CTX and THP on Foxp3 expression were examined by flow cytometry and real-time PCR assays.

RESULTSFlow cytometry analysis showed that LA-N-6 cells expressed Foxp3 at a high level. At sub-optimal concentration, chemotherapy drugs CTX and THP significantly down-regulated expression of Foxp3 on LA-N-6 cells at protein level (P<0.05). CTX also decreased the expression of Foxp3 at mRNA level (P<0.05). CONCLSUSIONS: Neuroblastoma cells LA-N-6 express Foxp3 at a high level, which can be suppressed by chemotherapy drugs CTX and THP. These data suggest that chemotherapy might suppress the growth and metastasis of tumor cells partially through inhibiting the expression of Foxp3.

Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cyclophosphamide ; pharmacology ; Doxorubicin ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Forkhead Transcription Factors ; analysis ; antagonists & inhibitors ; genetics ; Humans ; Neuroblastoma ; drug therapy ; immunology ; pathology ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effects of vasoactive intestinal peptide (VIP) on the airway inflammation and its regulatory effect on Th17/Treg imbalance in asthmatic mice.

METHODSA total of 30 BALB/c mice were equally and randomly divided into three groups: control, asthma, and VIP. An acute asthmatic mouse model was established by sensitization and challenge with ovalbumin (OVA). The control group received normal saline instead of OVA. Before the challenge with OVA, the VIP group was administered VIP (20 μg/mL) by aerosol inhalation for 30 minutes. The bronchoalveolar lavage fluid (BALF) and the lung tissue were collected from mice. The pathological changes in the lung tissue were observed by hematoxylin and eosin staining. The levels of Th17/Treg-related cytokines in BALF were measured by enzyme-linked immunosorbent assay. The expression of retinoid-related orphan receptor gamma t (RORγt) and forkhead box P3 (Foxp3) were measured by real-time fluorescence quantitative PCR and immunohistochemistry.

RESULTSThe histopathological results showed that the VIP group had milder symptoms of airway inflammation than the asthma group. The level of IL-17 in BALF in the asthma group was significantly higher than that in the control group and the VIP group (P<0.01), but the level of IL-17 in the control group was significantly lower than that in the VIP group (P<0.01). The level of IL-10 in BALF in the asthma group was significantly lower than that in the control group and the VIP group (P<0.01, but the level of IL-10 in the VIP group was significantly higher than that in the control group (P<0.01). The asthma group showed significantly higher expression levels of RORγt mRNA and protein in the lung tissue and significantly lower expression levels of Foxp3 mRNA and protein than the control group (P<0.01). The VIP group had significantly lower expression levels of RORγt mRNA and protein in the lung tissue and significantly higher expression levels of Foxp3 mRNA and protein than the asthma group (P<0.05).

CONCLUSIONSThe Th17/Treg imbalance may be closely related to the airway inflammation in asthmatic mice. VIP can improve airway inflammation by regulating the Th17/Treg imbalance in asthmatic mice.

Animals ; Asthma ; drug therapy ; immunology ; Forkhead Transcription Factors ; genetics ; Interleukin-10 ; analysis ; Interleukin-17 ; analysis ; Male ; Mice ; Mice, Inbred BALB C ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; genetics ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology ; Vasoactive Intestinal Peptide ; pharmacology ; therapeutic use