[Hepatitis B virus antigen peptide presentation by Epstein-Barr virus-transformed peripheral blood B cells].

Objective: To establish an Epstein-Barr virus-transformed peripheral blood B cell line (BCL), and explore its phenotypic characteristics, the ability to secrete antibodies and cytokines, and the ability to present hepatitis B virus (HBV) antigen peptide. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from patients with HBV infection. Epstein-Barr virus supernatant was incubated to construct BCL. The expression of CD19, CD138, CD38, CD27 and the production levels of IFN - γ, IL-10, IL-6 were detected by flow cytometry. BCL loaded with HBV antigen peptide was incubated with in vitro-expanded autologous T cells. Intracellular staining was used to detect the level of interferon-gamma produced by T cells. Results: Compared with untransformed peripheral blood B cells, BCL had high expression levels of CD138, CD38 and CD27, and the difference was statistically significant (P < 0.05), while the level of IL-6 production was decreased, and the difference was statistically significant (P < 0.01). BCL loaded with HBV antigen peptide had significantly enhanced the production of interferon-gamma by in vitro-expanded autologous T cells, and the difference was statistically significant (P < 0.01). Conclusion: BCL highly expresses CD138, CD38 and CD27, but its ability to produce IL-6 decreases. BCL can improve the immune response efficiency of HBV-specific T cells to HBV antigen peptide, and serve as a new tool for hepatitis B immune research.

Evaluation of clinical significance of endoglin expression during breast cancer and its correlation with ER and PCNA.

OBJECTIVE: The present study is aimed to investigate the expression of Endoglin during breast cancer. Moreover, its clinical pathological significance, as well as correlation with estrogen receptor (ER) and proliferating cell nuclear antigen (PCNA), were also studied. PATIENTS AND METHODS: qRT-PCR and Western blot assays were utilized to study PCNA mRNA, ER, Endoglin and protein expression. Immunohistochemistry analysis was conducted to determine the expression of Endoglin, ER, and PCNA protein in breast cancer tissue and adjacent cancer tissue. The microvascular density (MVD) was indicated by expression of Endoglin protein. The positive cell rate was used to express the protein expression level of ER and PCNA. RESULTS: The mRNA expression levels of Endoglin, ER, and PCNA were significantly elevated in breast cancer tumor tissues in comparison with the cancer-adjacent tissues. The positive expression rates of Endoglin, ER, and PCNA were 69%, 56%, and 73% respectively in breast cancer tumor tissues. Endoglin MVD values in breast cancer tissues and cancer-adjacent tissues were (35.18 ± 9.57)/mm2 and (7.21 ± 1.63)/mm2, respectively. The expression of Endoglin protein in breast tumor tissues was positively correlated to lymph node metastasis and TNM stages, but it was not to menopause and tumor size. Endoglin protein was positively correlated to the expression of PCNA protein, but was not correlated to ER expression. CONCLUSIONS: Endoglin protein expression is positively correlated to PCNA protein expression. So, Endoglin MVD in breast cancer tissues has important clinical significance in the assessment of breast cancer prognosis.

First Report of Fusarium equiseti Causing a Sheath Rot of Corn in China.

Corn is the most important cereal crop in China. Over 34.94 million ha of corn is cultivated in the country annually. However, fungal diseases are a major limiting factor in corn production. In August 2008, 50 ha in several corn fields in Liaoning, Jilin, and Heilongjiang provinces were observed to be severely affected by a disease causing a yield loss of 30%. Results from field surveys suggested an epidemic during late corn growth stages that affected corn sheaths, causing irregularly circular spots with grayish brown to dark brown lesions. Lesions ranged from 2.5 to 3 × 3 to 5 cm. To isolate the causal agent, tissue was removed from the border of lesions and surface sterilized in 75% ethanol for 30 sec and 0.1% HgCl2 for 1 min. The sample was then triple rinsed in sterile distilled water. The isolate was purified and subcultured on potato dextrose agar (PDA) at 25 ± 2°C. The initial color of the mycelium was white, turning brown after being cultured for 7 days. A pale brown to dark brown pigment developed in the agar beneath the colony. Chlamydospores, solitary but also in short chains, measuring 7.2 to 15.3 µm, were produced on carnation leaf agar (CLA) after 10 days and became verrucose 20 days later. Macroconidia were produced on CLA in orange sporodochia from monophialides on branched conidiophores, usually 5- to 7-septate, and apical cells were tapered and elongate. Basal cells were prominent, foot-shaped, and elongated in appearance. Microconidia were not observed (1). These morphological characteristics matched the description of Fusarium equiseti reported by Leslie and Summerell (1). A pathogenicity test was conducted with an isolate from each of the 36 corn plants by spraying 2 ml of spore suspension (106 conidia/ml) on 45-day-old corn sheaths (cv. Huang Zao). For the control treatment, 36 corn plants were sprayed with an equal volume of sterilized water. Inoculated plants were placed in a greenhouse at 32 to 34°C and 95% relative humidity. Typical irregularly circular lesions were observed 7 days after inoculation, except in the control samples. Each treatment was replicated three times. The suspected pathogen was consistently re-isolated from diseased tissue according to Koch's postulates, and was found to be morphologically similar to F. equiseti. Preliminary morphological identification of the fungus was confirmed by a PCR assay using genomic DNA extracted from the mycelia of a 7-day-old culture on PDA at 25 ± 2°C. A 750-bp amplified region of the transcription elongation factor (TEF) of rDNA was generated using TEF1 (5'-ATGGGTAAGGAGGACAAGAC-3') and TEF2 (5'-GGAAGTACCAGTGATCATGTT-3') primers. The TEF region (GenBank Accession No. KF754798) was sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) and displayed 99% nucleotide similarity with the rDNA-TEF of F. equiseti (JN127347.1) separately after a BLASTn search in GenBank. Based on the symptoms, fungal morphology, TEF sequence, and pathogenicity testing, this fungus was identified as F. equiseti. To our knowledge, this is the first report of F. equiseti on corn sheaths in China. This report will establish a foundation for further study of F. equiseti to address the disease effectively and to determine the severity of damage caused by F. equiseti. Reference: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006.

First Report of Bipolaris papendorfii Causing Corn Leaf Spot in China.

Corn is the most important cereal crop in China, with over 34.94 million ha being cultivated in the country annually. However, fungal diseases are a major limiting factor in corn production. In August 2012, 20 ha of corn fields in Anhui Province were found to be heavily infected by fungi. The margin of the lesion was achlorotic, and the middle was yellowish white or off-white, which was similar to the corn Curvalaria leaf spot. The oval lesions were approximately 5 to 7 mm. Lesion tissue was removed from the border between symptomatic and healthy tissue. The surface was sterilized in 75% ethanol for 30 s and 0.1% HgCl2 for 1 min, after which the sample was washed three times in sterile distilled water. The isolate was purified and subcultured on potato dextrose agar (PDA) at 25 ± 2°C. The initial color of the colony was light brown, turning dark brown after being cultured for 7 days. The conidia were boat-shaped or inverted pear-shaped and were clearly bent to one side. The cells of both ends were slightly lighter and respectively ranged from 34.5 to 44.0 µm and 12.0 to 21.0 µm away from the base, with the second cell as the widest. The majority conidia had three or four false septates; isolates produced light brown to medium brown conidiophore, scattered or clustered, often branching, and exhibited bending. These morphological characteristics matched with the description of Bipolaris papendorfii reported by Zhang (3). A pathogenicity test was conducted with the two isolates on each of the 36 corns by spraying 2 ml spore suspension (106 conidia/ml). For the control treatment, 36 corns were inoculated with an equal volume of sterilized water. Inoculated plants were placed in a greenhouse from 29 to 33°C and 95% relative humidity. The typical 5 to 7 mm oval lesions were observed 7 days after inoculation, except on the control samples. Three replications of 36 corns were used for each treatment. The isolate was consistently 100% reisolated from the diseased tissue according to Koch's postulate. The isolate was found to be morphologically similar to B. papendorfii. Preliminary morphological identification of the fungus was confirmed by PCR assay using genomic DNA extracted from the mycelium of a 7-day-old culture on PDA at 25 ± 2°C. A 550-bp amplified region of the internal transcribed spacer (ITS) of rDNA was generated using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') universal primers (1). The ITS region (GenBank Accession No. KC592365) was then sequenced by Sangon Biotech (Shanghai, China), and displayed 99% nucleotide similarity with the rDNA-ITS of B. papendorfii (JQ753972.1) separately after BLASTn research in GenBank. Based on the symptoms, fungal morphology, ITS sequence, and pathogenicity testing, this fungus was identified as B. papendorfii. The pathogen could reportedly infect tobacco and cotton (2). To our knowledge, this is the first study to report that B. papendorfii can infect corn in China. This report will establish a foundation for the further study of B. papendorfii to address the disease effectively. Further studies will be conducted to determine the incidence of the disease and the severity of damage caused by B. papendorfii as well as determine a possible mode for controlling the spread of the disease. References: (1) Y. J. Cao et al. Chin. J. Trop. Crops 31:1098, 2010. (2) H. Deng et al. Mycosystema 21:327, 2002. (3) T. Y. Zhang. Chin. Fungi Chi. 30:21, 2010.

[Regulation of traditional Chinese medicine formulae bailong on cAMP-PKA and DAG-PKC signal pathways of human gastric cancer cells].

OBJECTIVE: To evaluate the effects of traditional Chinese medicine formulae (Bailong) on cAMP/PKA and diacylaglycerol (DAG) protein kinase (PKC) pathways of the MGc80-3 cells. METHODS: cAMP level, DAG content and activities of PKA and PKC were measured in different groups: (1) control, (2) 1.8 mg/ml Bailong, (3) 1.8 mg/ml Bailong +20 ug/ml PKA inhibitor, (4) 5 mumol/L PKC inhibitor. RESULTS: When MGc80-3 cells were in treatment with Bailong for 3 hours, cAMP level and PKA activity were 11.27%, and 19.7% higher than that of the control, while DAG content and PKC activity were 47.0% and 64.2% lower than that of the control; When PKC pathway was blocked by PKC inhibitor GF-109203X, cAMP level and PKA activity were increased by 78.8% and 33.5% as against that of the control, while the DAG content and PKC activity were decreased by 40. 3% and 56.3% respectively. When MGc80-3cells were treated with Bailong, and at the same time, blocked PKA pathways by PKA inhibitor, cAMP level and PKA activity were decreased by 46.0% and 28.9%, on the other hand, DAG content and PKC activity were increased by 50.7% and 51.6% as against that of the Bailong group. CONCLUSIONS: (1) There was relationship of causes and result between differentiation of MGc80-3 cells and the signal pathways. (2) Results of this study were similar to that of hexamethylenebisacetamide (HMBA). It is shown that the two signal systems are the foundation of regulative effects of Chinese medicine formulae Bailong or HMBA on proliferation and differentiation in MGc80-3 cells.

Co-regulative effects of the cAMP/PKA and DAG/PKC signal pathways on human gastric cancer cells during differentiation induced by traditional Chinese medicines.

AIM: To evaluate the role of cAMP/PKA and DAG/PKC pathways of MGc80-3 cells treated with a traditional Chinese medicine compound, bailong preparation (bailong). METHODS: cAMP level, DAG content and activities of PKA and PKC were measured in different groups: control; 1.8 g/L bailong; 1.8 g/L bailong + 20 mg/L PKA inhibitor; and 5 µmol/L PKC inhibitor. RESULTS: When MGc80-3 cells were treated with bailong for 3 h, cAMP level and PKA activity were 113% and 19.7% higher than those of the control, while DAG content and PKC activity were 47.0% and 64.2% lower than those of the control. When the PKC pathway was blocked by PKC inhibitor GF-109203 X, cAMP level and PKA activity were increased by 78.8% and 33.5% compared to inhibitor GF-109203 X, and cAMP level and PKA activity were increased by 78.8% and 33.5% compared to the control, while the DAG content and PKC activity were decreased by 40.3% and 56.3%. When MGc80-3 cells were treated with bailong and PKA inhibitor blocked PKA pathways at the same time, cAMP level and PKA activity were decreased by 46.0% and 28.9%. On the other hand, DAG content and PKC activity were increased by 50.7% and 51.6% compared to the bailong group. CONCLUSION: There is a relationship of cause and effect between differentiation of MGc80-3 cells and the signal pathways. The results of this study are similar to that of hexamethylenebisacetamide (HMBA), suggesting that the two signal systems are the foundation of proliferative regulation of MGc80-3 cells treated with Chinese medicine bailong or HMBA.

The murine (C3H/He) epidermal Ia+ dendritic cells (Ia+DECs) and Thy-1+ dendritic cells (Thy-1+DECs) in contact hypersensitivity and aging.

The contact sensitivity evaluated by the ear swelling test and the dynamic changes of epidermal Ia+ dendritic cells (Ia+DECs) and Thy-1+ dendritic cells (Thy-1+DECs) were studied in trinitrochlorobenzene (TNCB) sensitized different age group C3H/He mice after challenge. A significant increase of ear swelling was observed between 6 h and 10 days of both 8-10 week (wk) and 40-48 wk groups; the ear swelling indices of 8-10 wk group were significantly higher than those of 40-48 wk group from 18 h to 5 days. A significant decrease of the densities of Ia+DECs from 18 h to 48 h, followed by a gradual increase reaching significant increase of the densities of Ia+DECs from 5 days to 21 days in both 8-10 wk and 40-48 wk groups, was observed; the densities of Thy-1+DECs significantly decreased from 18-48 h, followed by a gradual increase reaching a significant increase from 5 days to 21 days in both 8-10 wk and 40-48 wk groups. In the normal control groups, a significant decline of both Ia+DECs and Thy-1+DECs in the 40-48 wk group was observed. Results suggest that contact allergy may be diminished in aged mice. On the other hand, like Ia+DECs, Thy-1+DECs seem to be involved in the process of contact allergy.

Study on the E-rosette-blocking effects of E receptors.

Lymphocytes were purified from 10 samples of normal peripheral blood. E receptors were detached by heating to +45 degrees C. The sheep red blood cells (SRBC) and 2-aminoethylisothiouronium bromide (AET)-treated SRBC (AET-SRBC) were incubated at +4 degrees C for 2 h, with E receptors. E receptors detached by heating blocked E-rosette formation with SRBC, but not with AET-SRBC. The results suggest that E receptors may exhibit different conformational structures or different molecules which are responsible for E-rosette formation and E-AET-rosette formation, respectively.

Subpopulations of mononuclear cells in microscopic lesions of psoriatic patients. Selective accumulation of suppressor/cytotoxic T cells in epidermis during the evolution of the lesion.

The age of microscopic lesions in psoriatic subjects was assessed from the stacking characteristics in the horny layer and related to type and density (cells/tissue volume) of mononuclear cells in the epidermis and the dermis determined by immunoperoxidase methods using monoclonal antibodies. Pan T cells (Lyt-2+, Lyt-3+, Leu-4+, OKT3+), T helper cells (Leu-3a+, OKT4+), T suppressor/cytotoxic cells (Leu-2a+, OKT8+), Ia+ cells and monocytes (OKM2+, BRL alpha mono+) were determined in epidermis and dermis. The psoriatic lesion was divided into regions underneath a parakeratotic and an orthohyperkeratotic/hypergranular portion of the horny layer and contrasted with perilesional and uninvolved psoriatic skin as well as with healthy skin. In the various regions and skin layers, the cell density was highest in parakeratosis and decreased toward normality with decreasing histologic abnormality. The relation between epidermal and dermal cell densities of the T-cell subsets was modified in the involved psoriatic skin with a selective preponderance of T suppressor/cytotoxic cells in the epidermis. The accumulation was present in the youngest lesion found (3 days) and cell densities were unchanged in older lesions. The findings suggests that the altered relationship in the subsets of T cells has an important role during the induction and progress of the psoriatic process in the skin.

T lymphocyte subpopulations in alopecia areata and psoriasis: identification with monoclonal antibodies and Fc receptors.

A comparison of T lymphocyte subpopulations as defined by Fc receptors and monoclonal antibodies was investigated in 9 patients with alopecia areata and alopecia universalis (AA and AU) and in 6 patients with psoriasis. It was shown that there was higher proportion of T lymphocytes with Fc receptors for IgG (Tg cells) in patients with alopecia (AA and AU) and psoriasis. The proportions of total T lymphocytes (Tt), T lymphocytes with Fc receptors for IgM, T suppressor/cytotoxic cells (Leu2A), T helper/inducer (Leu3A) as defined by monoclonal antibodies were within normal range as compared to the normal donors. The possible reason of the dissociation between Tg and T suppressor (Leu2A) cells could be that these cells belong to different subpopulations.

T cell subpopulations and their functions in vitro. A study in patients with alopecia areata and alopecia universalis.

T lymphocyte subpopulations and their in vitro activities were determined in patients with alopecia areata (AA) and alopecia universalis (AU). The frequencies of T cells with receptors for IgG and T cells with receptors for IgM were determined in 16 cases. The IgG and IgM production of B lymphocytes cocultured with autologous and allogeneic T lymphocytes in the presence of PWM was determined in 12 and 9 cases, respectively, and the antibody-dependent cell-mediated cytotoxicity (ADCC) of unfractionated lymphocytes was examined in 6 cases. Patients with AA and AU had a higher proportion of Tg cells and more pronounced ADCC of unfractionated peripheral lymphocytes than normal persons. Furthermore, PWM-induced Ig production of B lymphocytes in the presence of autologous T lymphocytes seemed to be lower than that of controls.

T lymphocyte subpopulations and pokeweed mitogen-induced immunoglobulin synthesis in vitro by mononuclear cells from psoriasis patients.

T-lymphocyte subpopulations and pokeweed mitogen(PWM)-induced immunoglobulin (Ig) synthesis of mononuclear cells in vitro were studied in patients with psoriasis as well as in age- and sex-matched normal blood donors. The frequences of T cells with receptors for IgG (Tg) and T cells with receptors for IgM (Tm) were examined. The IgG and IgM synthesis in mononuclear cell suspensions in the presence of different amounts of PWM was determined. The percentage of total T cells (rosetting with neuroaminidase-treated sheep red blood cells), including high and low affinity T lymphocytes showed no difference between the patient group and controls. The patients with psoriasis had a significantly higher mean proportion of Tg cells than the normal donors whereas there was no significant difference in the proportions of Tm cells between these two groups. The PWM-induced Ig synthesis in the mononuclear cell suspension seemed lower in patients with psoriasis than in controls, the difference being statistically significant when the results were expressed as ratios of Ig amounts present in the supernatants of PWM-stimulated and non-stimulated cultures (index of stimulation).

Blood lymphocyte subpopulations in Mycosis fungoides and their functions in vitro.

T-lymphocyte subpopulations, PWM stimulated in vitro Ig production by mononuclear cells, and spontaneous cell-mediated cytotoxicity (SCMC) were determined in patients with Mycosis fungoides (MF) as well as in sex- and age-matched normal controls. We were able to confirm our earlier findings of a significantly higher frequency of T-lymphocytes with Fc-receptors for IgG (Tg cells) and a proportion of T lymphocytes with Fe receptors for IgM (Tm cells) not significantly differing from normal controls. As defined by monoclonal antibodies, the proportion of T-helper cells (Leu3A) was significantly lower, whereas the T-suppressors cells (Leu2A) were in the range of normal controls. The PWM stimulated and spontaneous Ig synthesis in vitro was lower in MF patients whereas there was no difference in the SCMC activity, in comparison with normal controls. The lower frequency of T-helper cells in the peripheral blood may be explained by their migration to the skin.

Spontaneous cell-mediated cytotoxicity (SCMC) in patients with alopecia universalis.

The spontaneous cell-mediated cytotoxicity (SCMC) of unfractionated lymphocytes was determined in 5 patients with alopecia universalis (AU). The target cells used were K562 and Chang cell lines. All patients had an increased SCMC as compared with age- and sex-matched donors. The results indicate that SCMC against K562 cells is more pronounced than that against Chang cells.

Blood lymphocyte subpopulations and antibody dependent, cell-mediated cytotoxicity (ADCC) in alopecia areata and universalis.

T-lymphocyte subpopulations and antibody-dependent, cell-mediated cytotoxicity (ADCC) were studied in patients with alopecia areata and universalis. The proportions of T-cells with the receptors for IgG (Tg) and T-cells with receptor for IgM (Tm) were determined in 16 cases. The ADCC of unfractionated lymphocytes was examined in 5 cases. A higher proportion of Tg-cells (T-cell with low affinity for sheep red cells) and a more pronounced ADCC of the peripheral lymphocytes were found in patients with alopecia areata and universalis than in age- and sex-matched normal donors.