Chemical Constituents and Molecular Mechanism of the Yellow Phenotype of Yellow Mushroom (Floccularia luteovirens).

(1) Background: Yellow mushroom (Floccularia luteovirens) is a natural resource that is highly nutritional, has a high economic value, and is found in Northwest China. Despite its value, the chemical and molecular mechanisms of yellow phenotype formation are still unclear. (2) Methods: This study uses the combined analysis of transcriptome and metabolome to explain the molecular mechanism of the formation of yellow mushroom. Subcellular localization and transgene overexpression techniques were used to verify the function of the candidate gene. (3) Results: 112 compounds had a higher expression in yellow mushroom; riboflavin was the ninth most-expressed compound. HPLC showed that a key target peak at 23.128 min under visible light at 444 nm was Vb2. All proteins exhibited the closest relationship with Agaricus bisporus var. bisporus H97. One riboflavin transporter, CL911.Contig3_All (FlMCH5), was highly expressed in yellow mushrooms with a different value (log2 fold change) of -12.98, whereas it was not detected in white mushrooms. FlMCH5 was homologous to the riboflavin transporter MCH5 or MFS transporter in other strains, and the FlMCH5-GFP fusion protein was mainly located in the cell membrane. Overexpression of FlMCH5 in tobacco increased the content of riboflavin in three transgenic plants to 26 µg/g, 26.52 µg/g, and 36.94 µg/g, respectively. (4) Conclusions: In this study, it is clear that riboflavin is the main coloring compound of yellow mushrooms, and FlMCH5 is the key transport regulatory gene that produces the yellow phenotype.

TRIM66 promotes malignant progression of hepatocellular carcinoma by inhibiting E-cadherin expression through the EMT pathway.

OBJECTIVE: The aim of this study was to explore the regulatory role of TRIM66 in the development of hepatocellular carcinoma (HCC), and to investigate its underlying mechanism. PATIENTS AND METHODS: A total of 88 pairs of HCC tissues and para-cancerous tissues were surgically resected. The expression of TRIM66 was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The correlation between TRIM66 expression and clinic-pathologic characteristics of HCC patients was analyzed. Follow-up data of enrolled HCC patients were collected for survival analysis. Subsequently, TRIM66 expression in HCC cells was determined by qRT-PCR as well. By constructing si-TRIM66, the biological performances of transfected HCC cells were determined using cell counting kit-8 (CCK-8), colony formation and transwell assay. Western blot was performed to measure the protein expressions of relative genes in epithelial-mesenchymal transition (EMT) pathway. Finally, HCC cells were co-transfected with si-TRIM66 and pcDNA-E-cadherin, followed by detection of invasive and migratory abilities. RESULTS: TRIM66 was highly expressed in HCC tissues compared with that of para-cancerous tissues. High expression of TRIM66 was positively correlated with tumor stage, lymph node metastasis and distant metastasis, whereas not correlated with age and sex of HCC patients. Kaplan-Meier curves revealed that a higher expression of TRIM66 was associated with worse prognosis of HCC. Similarly, TRIM66 was also highly expressed in HCC cells. The knockdown of TRIM66 in HCC cells significantly inhibited the proliferative, invasive and migratory abilities of transfected cells. However, TRIM66 down-regulation significantly induced cell apoptosis. Western blot results showed that TRIM66 knockdown in HCC cells markedly downregulated the protein expressions of E-cadherin, N-cadherin, Vimentin and ß-catenin. The inhibited migration and invasion of HCC cells resulted from TRIM66 knockdown were partially reversed by E-cadherin overexpression. CONCLUSIONS: TRIM66 is highly expressed in HCC, which is positively correlated with tumor stage, lymph node metastasis and distant metastasis of HCC patients. In addition, TRIM66 promotes the malignant progression of HCC by inhibiting E-cadherin through the EMT pathway.

MicroRNA-9-5p promotes osteoporosis development through inhibiting osteogenesis and promoting adipogenesis via targeting Wnt3a.

OBJECTIVE: To explore the role of microRNA-9-5p in regulating osteoporosis (OS) development and its underlying mechanism. PATIENTS AND METHODS: MicroRNA-9-5p expression in peripheral blood of 30 OS patients and 30 healthy subjects was examined by quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR). During the processes of osteogenesis and adipogenesis, mRNA levels of microRNA-9-5p, osteogenesis-related genes, and adipogenesis-related genes in marrow stromal stem cells (MSCs) were detected by qRT-PCR as well. After overexpression or knockdown of microRNA-9-5p, the regulatory effects of microRNA-9-5p on osteogenesis-related genes and adipogenesis-related genes in MSCs were accessed by detecting their mRNA and protein levels. Alizarin red staining and oil red staining were performed to determine the osteogenic and adipogenic capacities of MSCs after microRNA-9-5p overexpression, respectively. The dual-luciferase reporter gene assay was conducted to verify the binding condition of microRNA-9-5p and Wnt3a. Finally, rescue experiments were performed to confirm whether microRNA-9-5p could regulate OS development via targeting Wnt3a. RESULTS: Higher expression of microRNA-9-5p was found in OS patients than that of healthy controls. MicroRNA-9-5p expression was downregulated with the prolongation of osteogenic induction, whereas it was upregulated during the process of adipogenic differentiation. Overexpression of microRNA-9-5p downregulated mRNA levels of osteogenesis-related genes (ALP, RUNX2, and OPN), whereas upregulated adipogenesis-related genes (PPARγ, Adipsin, and C/EBPα) in MSCs. The number of calcified nodules became fewer after microRNA-9-5p overexpression in MSCs. MSCs that overexpressed microRNA-9-5p showed more lipid droplets than that of controls. Subsequently, the dual-luciferase reporter gene assay verified that Wnt3a is the target gene of microRNA-9-5p. Both mRNA and protein levels of Wnt3a were negatively regulated by microRNA-9-5p. Rescue experiments indicated that the regulatory effects of microRNA-9-5p on osteogenesis and adipogenesis of MSCs were reversed by Wnt3a overexpression. CONCLUSIONS: MicroRNA-9-5p is lowly expressed in the peripheral blood of OS patients. MicroRNA-9-5p promotes the occurrence and progression of OS through inhibiting osteogenesis and promoting adipogenesis via targeting Wnt3a.

MiR-7 regulates the PI3K/AKT/VEGF pathway of retinal capillary endothelial cell and retinal pericytes in diabetic rat model through IRS-1 and inhibits cell proliferation.

OBJECTIVE: To investigate the role of miR-7 in diabetic retinopathy and the underlying mechanism. MATERIALS AND METHODS: The rat model of diabetic retinopathy (DR) was established. After that, the endothelial cell (EC) and retinal pericyte (RP) were isolated. QRT-PCR was used to detect the expression of miR-7 and insulin receptor substrate-1 (IRS-1) in ECs and RPs cells while the protein level of IRS1 was detected by Western blot. miR-7 mimic and miR-7 inhibitor were transfected to achieve miR-7 overexpression or knockdown. Cell viability was detected by Cell Counting Kit-8 (CCK-8) assay after miR-7 overexpression or knockdown. Besides, the expression levels of PI3K, AKT, and VEGF were detected by Western Blot. The luciferase reporter assay was performed to investigate whether miR-7 could be combined with IRS-1. Conversely, whether miR-7 could affect IRS-1 was also verified. RESULTS: miR-7 expression was significantly decreased in ECs and RPs of the experimental group compared with the control group, while the mRNA and protein levels of IRS-1 were increased. The CCK-8 assay showed that overexpression of miR-7 decreased the cell activity in ECs and RPs. In contrast, knock-down of miR-7 could increase the cell viability. Besides, Western blot showed that after overexpression of miR-7, the expressions of PI3K, AKT, and VEGF in ECs and RPs cells were down-regulated. Meanwhile, miR-7 knockdown upregulated the protein levels of PI3K, AKT, and VEGF. The luciferase reporter assay suggested that the 3'UTR region of IRS-1 could be combined with miR-7, which may be the downstream target gene for miR-7. Moreover, knockdown of IRS-1 could reverse the effect of the miR-7 inhibitor on cell proliferation in the diabetic model. CONCLUSIONS: MiR-7 was lowly expressed in ECs and RPs cells. Overexpression of miR-7 can down-regulate the expression levels of PI3K, AKT, and VEGF by down-regulating its downstream target gene IRS-1, and ultimately inhibit the proliferation of retinal cells.

Exosomes miR-126a released from MDSC induced by DOX treatment promotes lung metastasis.

Acquired resistance to chemotherapy remains a major stumbling block in cancer treatment. Chronic inflammation has a crucial role in induction of chemoresistance and results, in part, from the induction and expansion of inflammatory cells that include myeloid-derived suppressor cells (MDSCs) and IL-13+ Th2 cells. The mechanisms that lead to induction of activated MDSCs and IL-13+ Th2 cells have not yet been identified. Here we demonstrated that doxorubicin (DOX) treatment of 4T1 breast tumor-bearing mice led to the induction of IL-13R+miR-126a+ MDSCs (DOX-MDSC). DOX-MDSC promote breast tumor lung metastasis through MDSC miR-126a+ exosomal-mediated induction of IL-13+ Th2 cells and tumor angiogenesis. The induction of DOX-MDSC is regulated in a paracrine manner. DOX treatment not only increases interleukin (IL)-33 released from breast tumor cells, which is crucial for the induction of IL-13+ Th2 cells, but it also participates in the induction of IL-13 receptors and miR-126a expressed on/in the MDSCs. IL-13 released from IL-13+Th2 cells then promotes the production of DOX-MDSC and MDSC miR-126a+ exosomes via MDSC IL-13R. MDSC miR-126a+ exosomes further induce IL13+ Th2 cells in a positive feed-back loop manner. We also showed that MDSC miR-126a rescues DOX-induced MDSC death in a S100A8/A9-dependent manner and promotes tumor angiogenesis. Our findings provide insight into the MDSC exosomal-mediated chemoresistance mechanism, which will be useful for the design of inhibitors targeting the blocking of induction of miR-126a+ MDSCs.

Overexpression of forkhead box protein M1 (FOXM1) plays a critical role in colorectal cancer

Background: The forkhead box M1 (FOXM1), an important regulator of cell differentiation and proliferation, is overexpressed in a number of aggressive human carcinomas. However, the clinical significance of FOXM1 signaling in human colorectal cancer (CRC) pathogenesis remains unknown. The aim of this study was to evaluate the role of FOXM1 in CRC tumorigenesis. Methods: We investigated FOXM1 expression in 103 cases of primary CRC and matched normal tissue specimens and explored the underlying mechanisms of altered FOXM1 expression and the impact of this altered expression on CRC proliferation and metastasis using in vitro models of CRC. Results: The results showed that high expression of FOXM1 staining was 85.44 % (88/103) in 103 cases of CRC and 20.39 % (21/103) in 103 cases of adjacent noncancerous tissue samples; the difference of FOXM1 expression between two groups was statistically significant (P < 0.001). Silencing of FOXM1 inhibited the proliferation of CRC cells, and the invasion and migration of CRC cells were distinctly suppressed. Furthermore, FOXM1 knockdown led to substantial reductions in VEGF-A levels in CRC cell lines. Conclusions: Our data suggest that the pathogenesis of CRC maybe mediated by FOXM1, and FOXM1 could represent selective targets for the molecularly targeted treatments of CRC

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Reproductive outcome of a case with familial balanced translocation t(3;6): implications for genetic counseling.

Although it is known that parental carriers of structural chromosomal rearrangements are associated with recurrent pregnancy loss, subsequent natural pregnancies remain possible. We examined the reproductive outcome of a familial balanced translocation with t(3;6)(q12;q27). Karyotyping of the proband revealed 46,XY chromosomes with the balanced translocation t(3;6). The first 2 pregnancies resulted in spontaneous abortions. Based on the proband karyotype, his father and half-brother were subjected to cytogenetic analysis, and both showed 46,XY, t(3;6)(q12;q27). After genetic counseling, the proband chose to continue the pregnancy. During the third pregnancy, the subject gave birth to a normal male infant. For parental carriers with balanced chromosomal translocations, natural pregnancy should be considered during genetic counseling.

Sperm retrieval from patients with nonmosaic Klinefelter's syndrome by semen cytology examination.

Successful sperm retrieval from ejaculates of nonmosaic Klinefelter's syndrome (KS) patients by using semen cytology examination was described in this report. The clinical parameters of KS patients with sperm compared to patients without sperm were described. One hundred and fifty-one patients were proven to suffer from KS by chromosomal analysis using G-banding. Spermatozoa were obtained from 10 patients (10/151, 6.6%) using semen analysis. After semen cytology examination, 32 patients (32/151, 21.2%) were found to have sperm or germ cell in their ejaculate. The patients with successful sperm retrieval were significantly younger (27.1 ± 3.7 years) than the patients for whom sperm retrieval failed (28.9 ± 4.2 years). The mean serum testosterone level and the mean T/LH ratio of KS patients with successful sperm retrieval were significantly higher in men with sperm than in men without sperm (testosterone: 3.2 ± 2.1 ng/mL vs 2.7 ± 1.5 ng/mL; T/LH ratio: 0.2 ± 0.3 vs 0.1 ± 0.1). In conclusion, semen cytology examination should be performed to identify sperm and germ cells in the ejaculate of KS patients if no sperm can be detected by traditional semen analysis. The serum testosterone level and T/LH ratio revealed an association between impaired Leydig cell function and impaired spermatogenesis in KS males. KS patients should receive earlier diagnosis and treatment.

Male infertility in Northeast China: molecular detection of Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome.

The prevalence of microdeletions of azoospermia factor (AZF) among azoospermic Klinefelter's syndrome (KFS) patients shows conflicting data. We aimed to detect this frequency in a Northeast Chinese population, and to investigate the possible association between AZF microdeletions and KFS by comparison with previous conflicting reports. Eighty men affected with KFS and a random healthy control group comprising 60 fertile men and women were recruited. AZF microdeletions were detected by multiplex polymerase chain reaction using 9 specific sequence-tagged sites. Karyotype analyses were performed on peripheral blood lymphocytes using standard G-banding. Finally, azoospermia was confirmed in 77 men affected with KFS and no AZF microdeletions were found. Karyotype analysis revealed 1 patient with karyotype 47,XXY,inv (9) (p11, q13), and 2 with mosaic karyotypes (46,XX/47,XXY and 46,XY/47,XXY). All other patients had karyotype 47,XXY. Review of the literature showed that these results were similar to those of other regions of Northeast Asia, but differed from those obtained from Caucasian populations. Our results supported the proposal that AZF microdeletions and KFS result from separate genetic defects. The prevalence of AZF in azoospermic KFS patients varies among populations, and it might result from genetic drift or selective pressure. These results suggest that routine screening for classical AZF microdeletions among infertile azoospermic men with a 47,XXY karyotype might not be necessary in Northeast Chinese individuals. However, it remains imperative for patients considering assisted reproductive treatments, particularly for those with mosaic karyotypes.

Diffusion-weighted MRI of the normal adult pancreas: the effect of age on apparent diffusion coefficient values.

AIM: To investigate and characterize the effect of age on apparent diffusion coefficient (ADC) values in the normal adult pancreas using diffusion-weighted magnetic resonance imaging (DWI). MATERIALS AND METHODS: Five hundred and fifty-nine adult patients without pancreatic disease, ranging from 20-81 years of age (mean 50.9 years; 436 men, 123 women), were included in this study. Breath-hold single-shot echo-planar DWI (b-values = 0, 500 s/mm(2)) was employed to determine the ADCs across all patients. Dependency of ADCs on age was characterized using a Spearman rank-order correlation test. RESULTS: Across the age spectrum, there was no significant correlation between ADC and age (p = 0.409). CONCLUSION: The findings of the present study suggest that the effect of age on ADCs can be excluded from the diagnosis of pancreatic diseases and design of future studies using breath-hold single-shot DWI and ADCs (as calculated with b-values of 0 and 500 s/mm(2)).

Exosomes and immune surveillance of neoplastic lesions: a review.

The immune system has been reported to suppress the development and progression of neoplastic lesions; however, the exact mechanisms by which neoplastic lesions and the immune system interact are not well understood. Within the last decade, tiny membrane bound particles, approximately 30-100 nm in diameter, have been observed in the blood and other body fluids. These particles, currently called exosomes, are released from many types of tissues including tumors, and they contain and carry many proteins, and mRNAs and microRNA species. We review here how tumors suppress the immune system, especially by the formation of exosomes. Exosomes released from tumors are carried in part by the vascular system to distant cells, which phagocytose them. Depending on the proteins, mRNAs or microRNAs in the exosomes and the cell type, phagocytosis of exosomes may provide a modulating signal to the cell. In the case of exosomes from tumors, uptake of the exosomes by cells of the immune system has been reported to have three main effects: 1) suppression of the number and activity of natural killer cells, 2) suppression of the activity of T cells and 3) suppression of the number and maturation of mature dendritic cells.

Ureaplasma urealyticum in male infertility in Jilin Province, North-east China, and its relationship with sperm morphology.

This study investigated the effects of Ureaplasma urealyticum infection and raised seminal leucocyte levels on sperm morphology in 967 infertile males and 201 fertile healthy volunteers. U. urealyticum infection led to a significant decrease in the percentage of morphologically normal sperm in infertile males. There was a clear correlation between U. urealyticum infection, raised seminal leucocytes and abnormal sperm morphology. The percentage of morphologically normal sperm was significantly lower in U. urealyticum-positive than U. urealyticum-negative infertile males or fertile controls. The percentage of morphologically normal sperm was lowest in U. urealyticum-positive males with raised seminal leucocytes. Previous studies have found raised seminal leucocyte levels to be associated with reactive oxygen species. The authors suggest that oxidative stress contributes to the effects of U. urealyticum on sperm morphology. In conclusion, U. urealyticum infection can negatively affect sperm morphology and this study provided two possible mechanistic explanations.

miR-155 promotes macroscopic tumor formation yet inhibits tumor dissemination from mammary fat pads to the lung by preventing EMT.

A micro-RNA, miR-155, is overexpressed in many types of cancer cells, including breast cancer, and its role(s) in tumor metastasis has been studied on a very limited basis. Tumor metastasis is a multi-step process with the last step in the process being formation of macroscopic tumor in organs distant from the primary tumor site. This step is the least studied. Here, we report that stable expression of miR-155 in 4T1 breast tumor cells reduces significantly the aggressiveness of tumor cell dissemination as a result of preventing epithelial-to-mesenchymal transition (EMT) of tumor cells in vivo. Further, miR-155 directly suppresses the expression of the transcription factor TCF4, which is an important regulator of EMT. However, when tumor cells are injected directly into the bloodstream, miR-155 remarkably promotes macroscopic tumor formation in the lung. Analysis of gene expression profiling identified a group of genes that are associated with promoting macroscopic tumor formation in the lung. Importantly, most of these genes are overexpressed in epithelial cells. Our findings provide new insight into how miR-155 modulates the development of tumor metastasis. This study suggests that the location of tumor cells overexpressing miR-155 is a critical factor: in mammary fat pads miR-155 prevents tumor dissemination; whereas in the lung miR-155 apparently maintains the epithelial phenotype of tumor cells that is critical for macroscopic tumor formation.

Seminal plasma zinc level may be associated with the effect of cigarette smoking on sperm parameters.

The aim of this study was to investigate the effect of cigarette smoking on seminal plasma zinc levels and sperm parameters, and to examine the role of seminal plasma zinc. Semen samples from 79 non-smokers and 68 smokers were obtained. There was a significant decrease in seminal plasma zinc in smokers and a clear correlation between seminal plasma zinc levels and the extent of smoking. Sperm parameters (concentration, motility and morphology) among smokers were significantly lower in comparison to non-smokers. These parameters were also significantly decreased among smokers with abnormal zinc levels, while there was no significant difference between non-smokers with normal zinc and non-smokers with abnormal zinc levels. As previous studies have shown that seminal plasma zinc is associated with a decrease of anti-oxidant defences, seminal plasma zinc could be a contributor to the effects of cigarette smoking on sperm parameters. In conclusion, cigarette smoking can affect sperm parameters and this study may help towards providing a mechanistic explanation.

TSPY is a cancer testis antigen expressed in human hepatocellular carcinoma.

In search for genes associated with hepatocellular carcinoma (HCC) by cDNA microarray, we found that the transcription of TSPY, 'testis-specific protein Y-encoded', was upregulated in HCC. Investigation of a broad spectrum of normal and malignant tissues by RT-PCR revealed the TSPY transcript selectively expressed in normal testis, different histological types of human neoplastic tissues, and tumour cell lines. The expression of TSPY in cancer cells was further confirmed by in situ hybridisation. Indirect immunofluorescence microscopy analysis showed that TSPY was localised mainly in the cytoplasm of transiently transfected cells. Testis-specific protein Y-encoded was detected in 50% (16 of 32) of well- and moderately differentiated HCC patients, in 16% (four of 25) of poorly differentiated HCC patients, and in 5% (one of 19) of renal cell cancer patients. A serological survey revealed that 6.6% (seven of 106) HCC patients had anti-TSPY antibody response, demonstrating the immunogenicity of TSPY in humans. In conclusion, these data suggest that TSPY is a novel cancer/testis (CT) antigen and may be a potential candidate in vaccine strategy for immunotherapy in HCC patients.

Defective clearance of adenovirus in IRF-1 mice associated with defects in NK and T cells but not macrophages.

A replication-defective adenovirus-LacZ recombinant virus (AdLacZ) was injected intravenously into IRF-1(-/-) mice and wild-type mice to characterize the contribution of IRF-1 to the immune-mediated clearance of Ad vector. Compared with wild-type mice, IRF-1(-/-) mice expressed higher levels of the LacZ gene product in the liver. After infusion of the AdLacZ, the expression of IRF-1 mRNA was upregulated in the liver of wild-type mice, but not in IRF-1(-/-) mice. Both spleen and liver mononuclear cells from IRF-1(-/-) mice initially exhibited a markedly lower number of NK, NK-T and CD8 T cells. At day 7 after the administration of AdLacZ, there was a significantly increased population of NK, NK-T and CD8 T cells in both spleen and liver, and also CD11b(+) cells in liver of IRF-1(-/-) mice, compared with the increased in wild-type mice. As IRF-1 is an important signal for production of IFN-gamma by CD8 T and NK cells as well as production of IL-12 by CD11b(+) cells, we determined whether there were lower levels of these cytokines in IRF-1(-/-) mice after Ad challenge. Surprisingly, there were lower levels of IL-12, but higher levels of IFN-gamma and IL-18 in IRF-1(-/-) compared with wild-type mice at day 7 after administration with AdLacZ. These results indicate that delayed clearance of Ad is associated with partial correction of defects of the NK, NK-T and CD8 T cells and increased production of IFN-gamma and IL-18 in IRF-1(-/-) mice.

Identification of multiple genetic loci that regulate adenovirus gene therapy.

A key aspect of the immune response to adenovirus (Ad) gene therapy is the generation of a cytotoxic T-cell (CTL) response. To better understand the genetic network underlying these events, 20 strains of C57BL/6 x DBA/2 (BXD) recombinant inbred (RI) mice were administered with AdLacZ and analyzed at days 7, 21, 30, and 50 for liver beta-galactosidase (LacZ) expression and CTL response. Sera levels of interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) were analyzed at different times after AdLacZ. There was a distinct strain-dependent expression of LacZ, which was strongly correlated with the CTL response. Among the five BXD RI strains that exhibited significantly prolonged LacZ expression, four also exhibited a marked defect in the production of Ad-specific CTL. There was a strong correlation between the sera levels of IFN-gamma, TNF-alpha, and IL-6, but cytokine responses were not significantly correlated with LacZ expression or the CTL response. Quantitative trait loci regulating LacZ on day 30 were found on chromosome (Chr) 19 (33 cM) and Chr 15 (42.8 cM). Cytotoxicity mapped to Chr 7 (41.0 and 57.4-65.2 cM), Chr 15 (61.7 cM), and Chr X (27.8 cM). IFN-gamma production mapped to Chr 18 (22, 27, and 32 cM) and Chr 11 (64.0 cM). TNF-alpha and IL-6 production mapped to Chr 6 (91.5 cM) Chr 9 (42.0 cM) and Chr 8 (52 and 73.0 cM). These results indicate that different strains of mice exhibit different pathways for effective clearance of AdLacZ depending on genetic polymorphisms and interactions at multiple genetic loci.

Functional supertype of HLA-A2 in the presentation of Flu matrix p58-66 to induce CD8+ T-cell response in a Northern Chinese population.

The functional supertype of HLA-A2 was investigated in the presentation of the A*0201-restricted Flu matrix p58-66 peptide to activate recall CD8+ T-cell response. In healthy Northern Chinese, the HLA-A2 supertype was mainly composed of the six alleles, A*0201 (26.4%), A*0206 (12.7%), A*0203 (8.2%), A*0207 (7.3%), A*0210 (1.8%) and A*0205 (0.9%), as analyzed by PCR using sequence specific primer (PCR-SSP) and sequence based typing (SBT). The IFN-gamma release Elispot assay was employed to assess effector CD8+ T cells. In A*0201-bearing individuals, the CD8+ T-cell response was potent when stimulated with autologous CD8- PBMCs. The frequency of the effector CD8+ T cells was 96.6% with the magnitude of effector CD8+ T cells of 225 SFC/5 x 104 CD8+ T cells and the RI of 25.7. In non-A*0201 individuals, the effector CD8+ T cells were minimally detectable while the peptide was presented by the autologous CD8- PBMCs. However, the induction of the response of CD8+ T cells obtained from non-A*0201 individuals was remarkably improved when the peptide was presented by autologous dendritic cells instead of CD8- PBMCs. The HLA-A2 alleles possessing cross-reactivity in the peptide presentation were mainly of A*0206 and non-A*0201 heterozygotes of A*0206 and A*0210. Moreover, A*0206 as the HLA-A2 functional supertype was further confirmed by tetramer assay. In two A*0206+ donors with CD8+ T-cell response to the peptide, the CD8+ T-cell frequency assessed by specific binding of peptide HLA-A*0201 tetramer was 4.62% and 1.66%, respectively. Thus, our results have substantiated the immunological relevance of the HLA-A2 supertype, which may benefit the design of peptide vaccines with the potential to be applicable in broader populations.

Age-related thymic involution in C57BL/6J x DBA/2J recombinant-inbred mice maps to mouse chromosomes 9 and 10.

A comprehensive analysis of initial thymus size and involution rate has not been quantitated for different genetic backgrounds of mice, thus genetic linkage analysis of thymic involution has not been possible. Here, we have used a mathematical method to analyze the age-related decline in thymocyte count in C57BL/6 and DBA/2 mice and have observed that thymic involution could be best fit with a negative exponential curve N(t)=beta(0) x exp(-beta(1)t), where t represents the age (day). This regression model was applied to C57BL/6 x DBA/2 (B x D) recombinant inbred strains of mice to identify the genetic loci influencing age-related thymic involution. There was a dramatic genetic effect of B and D alleles on thymocyte count at young age and the age-related thymic involution rate. The strongest quantitative trait loci (QTL) influencing the rate of thymic involution were mapped to mouse chromosome (Chr) 9 (D9Mit20 at 62 cM) and Chr 10 (D10Mit61 at 32 cM). The strongest QTLs influencing the initial thymocyte count were mapped to ChrX (DXMit324 at 26.5 cM) and Chr 3 (D3Mit127 at 70.3 cM). The present study suggests that the initial thymus size and the rate of thymic involution may be influenced by a relatively small number of genetic loci.

Cellular mechanism of thymic involution.

Involution of the thymus and alterations in the development of thymocytes are the most prominent features of age-related immune senescence. We have carried out a comparative analysis of thymocyte and stroma in rapid thymic involution DBA/2 (D2) strain of mice compared with slow involution C57BL/6 (B6) strain of mice. Analysis of mice at 15 months of age suggested an age-related decrease in the thymocyte cell count, a block in the development of T cells and cortical involution in D2 mice compared with 3-month-old mice. TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP-digoxigenin nick end labelling) staining and fluorescence-activated cell sorter (FACS) analysis showed that there was a significant increase in apoptotic cells in the cortex region of thymus in 15-month-old D2 mice compared with the same aged B6 mice. The thymocyte proliferation rate, as assessed by bromodeoxyuridine (BrdU) staining and [3H]-thymidine incorporation assay, was lower in 3-month-old D2 mice compared with the same age B6 mice. Immunohistochemical staining showed that the arrangement of MTS (mouse thymus stromal)-10+ epithelial cells and MTS-16+ connective tissue staining pattern had become disorganized in 15-month-old D2 mice but remained intact in B6 mice of the same age. These results suggest that, in D2 mice, both the thymocytes and stromal cells exhibit age-related defects, and that the genetic background of mice plays an important role in determining age-related alterations in thymic involution.