Electroacupuncture at Sensitized Acupoints Relieves Somatic Referred Pain in Colitis Rats by Inhibiting Sympathetic-Sensory Coupling to Interfere with 5-HT Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate whether electroacupuncture (EA) at sensitized acupoints could reduce sympathetic-sensory coupling (SSC) and neurogenic inflammatory response by interfering with 5-hydroxytryptamine (5-HT)ergic neural pathways to relieve colitis and somatic referred pain, and explore the underlying mechanisms.@*METHODS@#Rats were treated with 5% dextran sodium sulfate (DSS) solution for 7 days to establish a colitis model. Twelve rats were randomly divided into the control and model groups according to a random number table (n=6). According to the "Research on Rat Acupoint Atlas", sensitized acupoints and non-sensitized acupoints were determined. Rats were randomly divided into the control, model, Zusanli-EA (ST 36), Dachangshu-EA (BL 25), and Xinshu (BL 15) groups (n=6), as well as the control, model, EA, and EA + GR113808 (a 5-HT inhibitor) groups (n=6). The rats in the control group received no treatment. Acupuncture was administered on 2 days after modeling using the stimulation pavameters: 1 mA, 2 Hz, for 30 min, with sparse and dense waves, for 14 consecutive days. GR113808 was injected into the tail vein at 5 mg/kg before EA for 10 min for 7 consecutive days. Mechanical sensitivity was assessed with von Frey filaments. Body weight and disease activity index (DAI) scores of rats were determined. Hematoxylin and eosin staining was performed to observe colon histopathology. SSC was analyzed by immunofluorescence staining. Immunohistochemical staining was performed to detect 5-HT and substance P (SP) expressions. The calcitonin gene-related peptide (CGRP) in skin tissue and tyrosine hydroxylase (TH) protein levels in DRG were detected by Western blot. The levels of hyaluronic acid (HA), bradykinin (BK), prostaglandin I2 (PGI2) in skin tissue, 5-HT, tryptophan hydroxylase 1 (TPH1), serotonin transporters (SERT), 5-HT 3 receptor (5-HT3R), and 5-HT 4 receptor (5-HT4R) in colon tissue were measured by enzyme-linked immunosorbent assay (ELISA).@*RESULTS@#BL 25 and ST 36 acupoints were determined as sensitized acupoints, and BL 15 acupoint was used as a non-sensitized acupoint. EA at sensitized acupoints improved the DAI score, increased mechanical withdrawal thresholds, and alleviated colonic pathological damage of rats. EA at sensitized acupoints reduced SSC structures and decreased TH and CGRP expression levels (P<0.05). Furthermore, EA at sensitized acupoints reduced BK, PGI2, 5-HT, 5-HT3R and TPH1 levels, and increased HA, 5-HT4R and SERT levels in colitis rats (P<0.05). GR113808 treatment diminished the protective effect of EA at sensitized acupoints in colitis rats (P<0.05).@*CONCLUSION@#EA at sensitized acupoints alleviated DSS-induced somatic referred pain in colitis rats by interfering with 5-HTergic neural pathway, and reducing SSC inflammatory response.

Expression Level of MiR-146a in Acute Myeloid Leukemia Patients and Its Clinical Significance / 中国实验血液学杂志

OBJECTIVE@#To detect the expression of miR-146a in patients with AML, and to evaluate the relationship between miR-146a expression level and clinical characteristics, treatment response, EFS and OS.@*METHODS@#154 patients with newly diagnosed AML were enrolled in AML group, 50 controls (patients with thrombocytopenic purpura or voluntary donor of bone marrow) were enrolled in control group. The miR-146a expression levels in bone marrow mononuclear cells was detected by RT-PCR between 2 group. AML patients were treated with chemotherapeutic drugs, and their clinical response and survivals were assessed.@*RESULTS@#The expression level of MiR-146a in AML group was significantly lower than that in control group. The ROC showed that miR-146a could distinguish the patients in AML and control group better (area under curve 0.819 (95%CI: 0.761-0.877). Meanwhile, the proportion of good and moderate good prognosis (P＜0.001), proportion of WBC count ≤15.2×10/L (P＜0.05), CR rate (P＜0.05), EFS (P＜0.01) and OS (P＜0.01) in patients with high miR-146a expression were higher than those in patients with low miR-146a expression. Cox's model showed that miR-146a expression level positively realated with incressed EFS and OS.@*CONCLUSION@#MiR-146a is downregulated in AML patients, which might be served as a biomarker for predicting risk and prognosis of AML patients.

Differentially expressed genes in heads and tails of Angelica sinensis diels: Focusing on ferulic acid metabolism / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the scientific connotation of the discrepant pharmaceutical activities between the head and tail of Angelica sinensis diels (AS), an important herb extensively utilized in Chinese medicine, by the approach of transcriptome sequencing.</p><p><b>METHODS</b>Ten samples of AS were randomly collected in Min County, Gansu Province of China. Transcriptome sequencing of AS was accomplished in a commercial ILLumina HiSeq-2000 platform. The transcriptome of each head and tail of AS were fixed in a gene chip, and detected under the procedure of Illumina HiSeq-2000. Differentially expressed unigenes between the heads and tails of AS were selected by Shanghai Biotechnology Corporation (SBC) online analysis system, based on Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and relevant bioinformatic database.</p><p><b>RESULTS</b>Totally 63,585 unigenes were obtained from AS by high-throughput sequencing platform. Among which 3359 unigenes were identified as differentially expressed unigenes between the heads and tails of AS by SBC analysis system scanning. Of which 15 differentially expressed unigenes participate in the metabolic regulation of phenylpropanoid biosynthesis (PB) pathway and ferulic acid metabolites, in response to the distinguished pharmaceutical actions of the heads and tails of AS.</p><p><b>CONCLUSION</b>Different content of ferulic acid in the heads and tails of AS is related to the differentially expressed genes, particularly involved in the PB pathway.</p>

Tumor infiltrating regulatory T cells in human breast cancer and associated draining lymph nodes: an in-situ analysis / 中华病理学杂志

<p><b>OBJECTIVE</b>To retrospectively analyze the quantity and status of the tumor infiltrating regulatory T lymphocytes in breast cancer and the draining lymph nodes, and to elucidate the clinical pathologic significance.</p><p><b>METHODS</b>Seventy-four breast cancer samples with excised axillary lymph nodes were typed and staged histopathologically. The regulatory T lymphocytes were labeled by immunohistochemistry using EnVision method with the monoclonal antibodies against CD25 and Foxp3, and the immunophenotype was analyzed. In addition, the expression of IFN-γ, IL-10 and TGF-β1 mRNA in lymphocytes of lymph nodes draining the tumors was detected by in situ hybridization with the corresponding specific oligo nucleaic acid probes.</p><p><b>RESULTS</b>The number of CD25(+)Foxp3(+) T cells infiltrating the interstitium was much higher than that in the parenchymal tissue of the cancer. In the tumor draining lymph nodes, CD25(+) cells and Foxp3(+) cells were predominantly distributed in the paracortex with a proliferative pattern. TGF-β1, INF-γ and IL-10 mRNA positive cells showed a similar distribution pattern in the draining lymph nodes. Among the 39 cases with metastatic disease, the lymph nodes with metastases showed a much higher number of CD25(+)Foxp3(+) cells than that without metastases (23.5 vs 17.3 and 23.8 vs 15.5; P < 0.05). However, there was no difference in the density of Foxp3(+)CD25(+) cells in the draining lymph nodes between the death and survival groups (P > 0.05). Cytokine expression of TGF-β1, IL-10 and IFN-γ mRNA in the lymphocytes of draining lymph nodes in 24 cases showed that there were more IL-10 mRNA positive cells in the dead patients than that in the survived patients. A similar trend was observed for TGF-β1 mRNA positive cells but the difference was not statistically significant (P > 0.05). The expression rate of TGF-β1 and IL-10 mRNA in the draining lymph nodes was proportional to that of CD25(+) and Foxp3(+) cells (P < 0.05), and the expression of TGF-β1 positive cells was also proportional to that of IL-10 mRNA positive cells (P < 0.01). The expression of IFN-γ mRNA among these groups showed no significance (P > 0.05).</p><p><b>CONCLUSIONS</b>Regulatory T cells may play important roles in inhibiting the host antitumor immunity, and the presence of increased regulatory T cells and Th2-secreting cells in paracortex with a proliferative pattern in the tumor draining lymph nodes implies that the paracortical proliferation of draining lymph nodes may not reflect positive antitumor effects.</p>

Clinical study of standard dose of rituximab for the treatment of refractory primary immune thrombocytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of rituximab on B-lymphocytes and anti-platelet glycoprotein-specific antibodies in patients with refractory primary immune thrombocytopenic (ITP).</p><p><b>METHODS</b>Thirty-one ITP patients with a median age of 36 years (range 16 - 56 years) received solely intravenous rituximab at the dose of 375 mg/m(2) once weekly for consecutive 4 weeks. Lab studies included complete blood count, serum concentrations of IgG, IgM and IgA. CD3(+), CD4(+), CD8(+), CD19(+) and CD20(+) cell numbers were assayed by flow cytometry and anti-platelet glycoprotein-specific antibodies (GPIIb/IIIa, GPIb/IX) were assayed by monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) prior to and following rituximab therapy. The response was evaluated according to the response criteria of international working group of ITP.</p><p><b>RESULTS</b>Complete responses were achieved in 12 cases, response in 7 cases, and no response in 12 cases. Responses were sustained 2 to 28 months (median 6 months) with 4 cases relapsed. After 4 weeks of rituximab therapy, GPIIb/IIIa and GPIb/IX disappeared in responded patients, and CD 19(+)/CD20(+) cells were almost depleted in all patients. As expected, the serum concentrations of IgG, IgM, IgA, and the T cell counts were not changed after therapy. Four patients developed infusion-related reaction, 1 impaired renal function, and 3 secondary infections.</p><p><b>CONCLUSION</b>Rituximab is effective and safe, and the adverse reaction is tolerable.</p>

Clinicopathological significance of cytotoxic lymphocytes in breast cancer and draining lymph nodes / 中华病理学杂志

<p><b>OBJECTIVE</b>To analyze retrospectively the quantity and activation status of the tumor infiltrating cytotoxic lymphocytes in breast cancer and the draining lymph nodes, and its relation to the clinical pathological significance.</p><p><b>METHODS</b>Seventy-four breast cancer samples with their corresponding axillary lymph nodes were histologically typed and staged. Cytotxic lymphocytes were analyzed by immunohistochemistry with the monoclonal antibodies against CD8, CD56, granzyme B and perforin.</p><p><b>RESULTS</b>The number of infiltrating CD8(+) T cells in the cancerous interstitial tissue were much higher than that in the tumor parenchyma. Compared with the metastatic tumor samples, the CD8(+) T cells were more intensive in the primary tumors (35.7 +/- 16.0 vs. 23.7 +/- 9.6). The tumor infiltrating CD8(+) T cells of patients with 5 years survivals were more than that of the dead cases in this follow-up series death (32.9 +/- 14.1 vs. 20.1 +/- 9.9). There was no significant difference of activated tumor infiltrating cytotoxic T cell analyzed by using the activation marker granzyme B(+) and there was also no significant correlation between the intensity of CD8(+), CD56(+) cells and the clinicopathological stages. However, percentages of the activated cytotoxic lymphocytes in Stage I groups were significantly higher than those in stage III and IV. Moreover, the number of perforin(+) cells was significantly less than that of granzyme B(+) cells, particularly in the cancerous tissue, indicating a dysfunctional status of tumor infiltrating cytotoxic lymphocytes.</p><p><b>CONCLUSIONS</b>Activated cytotoxic lymphocytes may play a significant role against the tumor progression and is associated with a favorable prognosis to some extent. However, a putative dysfunctional status of cytotoxic lymphocytes at tumor site may compromise the host immunity against cancer.</p>

Preparation and activity detection of chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1.</p><p><b>METHODS</b>Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1.</p><p><b>RESULTS</b>After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells.</p><p><b>CONCLUSION</b>High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.</p>

Identification of metastasis-associated proteins of ovarian cancer by proteomics / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the differenal protein expression profiles of ovarian tumor cell lines with distinct metastatic abilities.</p><p><b>METHODS</b>The ovarian cancer cell line HO8910 and HO8910pm, derived from same parental cells but exhibited different metastatic ability, were investigated by two-dimensional gel electrophoresis (2-DE)-MALDI-TOF-MS proteomic approach.</p><p><b>RESULTS</b>Thirty-nine proteins were detected by 2-DE to have expression disparity levels over 2 folds between two cell lines. Eighteen of them were identified by MALDI-TOF-MS. The proteins are involved in apoptosis, extra cellular matrix (ECM), cytoskeleton, growth factor, glycolysis, protein metabolism and immune system.</p><p><b>CONCLUSION</b>The data are valuable for the identification of differentially expressed proteins involved in the biological behavior of human ovarian cancer.</p>

Over-expression of extracellular signal-regulated kinase in vascular smooth muscle cell of hypertensive rats / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.</p><p><b>METHODS</b>Two-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery. The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively. The control group were sham operated age-matched Wistar rats. Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.</p><p><b>RESULTS</b>Blood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198. 00 +/- 33. 00 mm Hg at the end of experiment, significantly higher than that in the control rats (P < 0.01). Blood pressure in SHR4w (108.00 +/- 11.25 mm Hg) was similar to that in the controls. However, it rose to 122.25 +/- 21.75 mm Hg in SHR8w, and even up to 201.75 +/- 18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P < 0.01). The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P < 0.05). Hyaline degeneration of the afferent arterioles was found in WHR. In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w. Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2. The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09% +/- 1.75%, 14.57% +/- 4.58%, 29.44% +/- 7.35%, and 13.63% +/- 3. 85%, respectively) than that of the controls(P < 0.01). The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09% +/- 1.40%, 24.17% +/- 6.92%, 32.44% +/- 4.05%, and 18.61% +/- 3.35%, respectively) than that of the controls (P < 0.01), too. The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P < 0.01).</p><p><b>CONCLUSION</b>Extracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR. Phospho-ERKI/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.</p>

P21ras and phosphorylated extracellular signal-regulated protein kinase are highly expressed in vascular smooth muscle cells of hypertensive rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms of vascular smooth muscle cell (VSMC) hyperplasia and extracellular signal-regulated protein kinase 1/2 (ERK 1/2) phosphorylation of VSMCs under the condition of hypertension.</p><p><b>METHODS</b>Wistar rat models of two kidney-one clip hypertension (2K1C) were established and their right kidneys were harvested 4 weeks after the operation. Immunohistochemistry and Western blotting were performed to detect phospho-ERK1/2 and P21ras protein expression in the VSMCs of the renal arterioles, and the results were compared with those from 16-week-old spontaneous hypertension rats (SHR16) and control rats.</p><p><b>RESULTS</b>The blood pressure of 2K1C Wistar rats was significantly increased from 104-/+18 mmHg to 198-/+33 mmHg at the end of the experiment, and the blood pressure of SHR16 reached 163-/+23 mmHg, significantly higher than that of the control rats (P<0.01). Compared with the control group, 2K1C rats showed obvious glomerular fibrosis (P<0.05) with hyaline degeneration of the afferent arterioles. In contrast, neither glomerular fibrosis nor hyaline degeneration of arterioles, nor protein cast was observed in SHR16. In 2K1C rats and SHR16, the positivity rates of phospho-ERK1/2 and p(21ras) staining in the VSMCs of the afferent arterioles and the interlobular, interlobar and arcuate arteries was significantly higher than those of the controls (P<0.01), and the expression of phospho-ERK1/2 and P(21ras) protein in the kidney was also significantly higher as revealed by Western blotting (P<0.01).</p><p><b>CONCLUSION</b>High expression of ERK1/2 and P(21ras) in the renal arteriole VSMCs of 2K1C hypertensive rats and SHR may play an important role in VSMC hypertrophy and proliferation in hypertension.</p>

New strategy of cancer immunotherapy: irradiation or chemotherapeutics-induced lymphopenia combined with immune reconstitution and tumor vaccine / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To test whether vaccination performed during irradiation or chemotherapeutics-induced lymphopenia-driven T cell proliferation could augment the antitumor immunity.</p><p><b>METHODS</b>The study composed of two parts, investigating the anti-tumor efficacy of performing tumor vaccination during early immune reconstitution period following sublethal total body irradiation and cyclophosphamide (Cy)-induced lymphopenia, respectively. Mice were subsequently reconstituted with naïve splenocytes from syngeneic mice and were named RLM (Reconstituted lymphopenic mice). Immunization/vaccination (F10) and adoptive immunotherapy (D5-G6) were used to explore anti-tumor immune responses in vaccinated irradiation/RLM and vaccinated Cy/RLM, respectively. Both normal C57BL/6 mice and RLM were vaccinated with irradiated, weakly immunogenic F10 melanoma cells and subsequently challenged with F10 cells. In addition, to determine the role of CD4(+) and CD8(+) T cells in the protective anti-tumor immune response, irradiation/RLM were depleted of these subpopulations by administration of the appropriate mAb around challenge. In the second part, adoptive immunotherapy was used to evaluate the anti-tumor immune responses under chemotherapeutics-induced lymphopenic condition. Both normal mice and RLM (Cy-treated) were vaccinated with GM-CSF-modified D5 melanoma cells (D5-G6) and tumor vaccine draining lymph nodes (TVDLN) were harvested 9-10 days later. Effector T cells were generated in vitro from TVDLN cells and adoptively transferred to mice bearing 3-day pre-established pulmonary metastases (D5). Recipient mice were sacrificed 2 weeks later after tumor inoculation and pulmonary metastases were enumerated.</p><p><b>RESULTS</b>Significantly greater protection was induced in vaccinated irradiation/RLM, compared to vaccinated normal mice (63.2% vs 16.7%). Protective immunity in RLM depended on CD8(+) T cells. Increase in the interval between reconstitution and vaccination significantly decrease the protection. Effector T cells generated from vaccinated Cy-treated RLM demonstrated significantly higher in vivo anti-tumor efficacy over those of vaccinated normal mice.</p><p><b>CONCLUSION</b>This study suggests that vaccination of RLM could elicit augmented antitumor immunity compared to normal hosts, highlighting the potential clinical benefit of performing tumor vaccination during irradiation or chemotherapeutics-induced lymphopenia in cancer patients.</p>

Expression of IgVH and B7-1 in proteome of the human colorectal carcinoma cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To conduct a proteomic analysis of human colorectal carcinoma cell lines LS174T and SW480 by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).</p><p><b>METHODS</b>The total proteins of human colorectal carcinoma cell lines LS174T and SW480 were separated with 2-DE using immobilized pH gradient strips and analyzed by MALDI-TOF-MS to obtain peptide mass fingerprints (PMFs). Proteins were identified by using Mascot software to search protein databases.</p><p><b>RESULTS</b>Good resolution 2-DE maps were obtained. Some proteins including immunoglobulin heavy chain variable region (IgVH) and co-stimulatory molecule B7-1 were identified. IgVH and B7-1 were confirmed by electrospray ionization tandem spectrometry (ESI-MS/MS) and immunocytochemistry.</p><p><b>CONCLUSION</b>There are IgVH and B7-1 expressions in human colorectal carcinoma cell lines LS174T and SW480. Results obtained will help to elucidate the mechanisms of tumor immune escape.</p>

Humoral immune response and in vitro neutralizing antibody assay on co-delivery of protein HPV16L1 virus-like particle with HPV16L1 gene / 中华病理学杂志

<p><b>OBJECTIVE</b>To compare humoral immune response by co-inoculating mice with antigen HPV16L1 virus-like particle (VLP) and HPV16L1 recombinant plasmids and then observing the neutralizing antibody activity in vitro.</p><p><b>METHODS</b>C57BL/6 mice were injected intramuscularly/subcutaneously with pcDNA-L1 plasmids plus HPV16L1 VLP. Serum IgG levels were detected by ELISA, antibody neutralizing protective activities were determined by hemagglutination inhibition and HPV16L1 VLP binding inhibition assay.</p><p><b>RESULTS</b>Serum antibody titers and neutralizing antibody activities were increased in HPV16L1 plasmids plus HPV16L1 VLP proteins in co-immunized mice when compared with controls.</p><p><b>CONCLUSION</b>Co-inoculation of the HPV16L1 VLP protein can enhance production of neutralizing antibody activities against aimed antigen, which should be a more promising strategy for effective HPV16 prophylactic vaccine development.</p>

Poorly-immunogenic tumor is capable of inducing proliferation of CD4 (+) CD25 (+) regulatory T-lymphocytes in vitro / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the distribution of regulatory T-lymphocytes in the splenocytes cocultured with syngeneic low-immunogenic tumor cells, as compared with that of highly-immunogenic tumor cells, to investigate the mechanism underlining tumor evasion.</p><p><b>METHODS</b>Three different immunogenic tumor cells were cocultured with syngeneic splenocytes individually to mimic cancer immunity in vitro. The proliferation response of splenocytes was measured by thymidine incorporation. The distribution of TR cells, CD4(+) IFN-gamma (+) T cells and CD4(+) IL-10(+) T cells were analyzed by flow cytometry. The secretion of IFN-gamma and IL-10 in supernatants was measured by ELISA assay.</p><p><b>RESULTS</b>The stimulation Index of splenocytes cocultured with syngeneic highly-immunogenic H22 or FBL3 was much higher than that of poorly immunogenic melanoma D5. In each group, stimulation Index of splenocytes cocultured with allogeneic tumor cells was higher than that of the corresponding tumor immunity model. In addition, compared with those of highly-immunogenic tumors, there were more TR, CD4(+)IL-10(+) and less CD4 (+)IFN-gamma(+) T cells in the splenocytes, and higher IL-10 and lower IFN-gamma levels in the supernatant of the splenocytes stimulated with low-immunogenic D5 cells.</p><p><b>CONCLUSION</b>Poorly-immunogenic tumor cells can induce the proliferation of TR cells, which may play an important role in tumor evasion.</p>

Mechanism of augmented anti-tumor immunity in reconstituted lymphopenic mice immunized with melanoma vaccine / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore mechanisms of the augmented anti-tumor immunity observed in reconstituted lymphopenic mice (RLM) receiving melanoma vaccination.</p><p><b>METHODS</b>The study is to investigate the anti-tumor immunity of tumor vaccination during early immune reconstitution period following irradiation and cyclophosphamide (CY)-induced lymphopenia. Lymphopenic mice were subsequently reconstituted with naive splenocytes from syngeneic mice and immunized with irradiated melanoma cells F10 (irradiation experiment) and GM-CSF-modified D5 melanoma cells (D5-G6) (CY experiment). Controls included normal C57BL/6 mice receiving the corresponding vaccination, un-immunized naive mice and RLM. 8 - 10 days after vaccination, tumor vaccine draining lymph nodes (TVDLN) were harvested and phenotyped by FACS analysis. T cells purified from TVDLN were stimulated with anti-CD3 and anti-TCRbeta and proliferation was assessed by [(3)H]-TdR incorporation and FACS assay was performed for CD69 expression.</p><p><b>RESULTS</b>The augmented anti-tumor immunity correlated with a significant increase in the percentage of T cells with activation/memory phenotype in the TVDLN of vaccinated RLM, compared to that of the controls. There was also a significant increase in the density of DCs in TVDLNs. The activation threshold of T cells generated from vaccinated RLM was significantly decreased, resulting in markedly enhanced proliferating capability upon anti-CD3 stimulation.</p><p><b>CONCLUSION</b>This study suggests that the augmented anti-tumor immunity observed in vaccinated RLM is due to down regulated activation threshold of T cells during lymphopenia-driven T cell proliferation, which may in turn facilitate the breaking down of immune tolerance to weak tumor antigens upon vaccination with tumor cell vaccines.</p>

Carboxyl terminus truncated HPV58 virus L1 protein expressed with baculovirus system and its bioactivity / 生物工程学报

To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine.

Preliminary study of HPV16 L1/E6-E7 chimeric recombinant DNA vaccine plasmid construction and expression in CHO cell / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To develop HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines.</p><p><b>METHODS</b>The nucleotides within HPV 16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mega primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV 16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV 16 L1 and E6 specific monoclonal antibodies.</p><p><b>RESULTS</b>ELISA showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were greater than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells.</p><p><b>CONCLUSIONS</b>Successful constructions of prophylactic and therapeutic DNA vaccine plasmids may be useful for future animal experiment and clinical trial.</p>

Genetic polymorphism of 3 STR loci of CSF1PO, TPOX and TH01 in Chinese Sibo population / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To obtain allele and genotype frequencies and related forensic data of CF1PO, TPOX and TH01 loci in Chinese Xinjiang Sibo population.</p><p><b>METHODS</b>Genomic DNA from peripheral blood mononuclear cells of normal Chinese Xinjing Sibo population was used as template, and CSF1PO, TPOX and TH01 fragments were amplified by PCR. The PCR products were analyzed by 4% denaturing PAGE and detected using silver stain detection.</p><p><b>RESULTS</b>Nine alleles were found at CSF1PO locus, eight alleles at TPOX locus and eight alleles at TH01 locus in Chinese Sibo population. All the 3 loci complied with Hardy-Weinberg equilibrium. The heterozygosities were 0.9426, 0.8361 and 0.8853, and the polymorphism information contents were 0.8298, 0.7213 and 0.7626 for CSF1PO, TPOX and TH01, respectively.</p><p><b>CONCLUSION</b>The data on the alleles frequency of these 3 STR loci might be used for individual identification and paternity identification and for genetic researches in Chinese Sibo population.</p>

Study of alginate/chitosan microcapsules for immobilization of Escherichia coli DH5 alpha / 生物工程学报

Objective proteins synthesized from genetically recombined Escherichia coli strain (E. coli) have been successfully produced by microbe fermentation, but complicated separation and purification steps always make against the maintenance of activities as well as increase the cost. Aiming at simplifying the process, an idea of administrating directly the microencapsulated genetically recombined E. coli is proposed. In this paper, study on culture of E. coli DH5 alpha immobilized in alginate/chitosan (ACA) microcapsule is presented. It was found that E. coli DH5 alpha grew well in the microcapsule with stable growth period longer than that of suspension culture, and cell aggregation phenomenon was observed. In vivo experiments showed that ACA microcapsules with E. coli DH5 alpha stayed over 48 h in mouse intestine, and the morphology of microcapsules was kept intact. These preliminary results have demonstrated that administration of microencapsulated E. coli DH5 alpha is safe, which laied the foundation for microencapsulated genetically recombined E. coli as carriers of gene engineering drugs.