Objective To determine the incidence, clinical manifestation and part of lymphokines which represent the balance of Th1 and Th2 in the role of the immunologic mechanisms for IRIS(immune restoration inflammatory syndromes)in patients initiating HAART(Highly Active Antiretroviral Therapy).Methods A prospective study of all patients initiating HAART was performed. A period of six months tracking initiating HAART was performed. The incidence of IRIS, time of occurrence and clinical disease spectrum were recorded. The main T lymphokines including IL-2, INF-γ, IL-4, IL-10 which on behalf of the balance of Th1 and Th2 were detected. To explore the immunopathologic mechanisms for IRIS, the levels of T lymphokines at pre-HAART, initiating HAART for 1 month, 3months and 6 months were compared in IRIS group and non-IRIS group, healthy group. Results A total of 212 patients were enrolled in this study. 59 patients were diagnosed as IRIS at a median of 21 days after HAART initiation (QR 19 days).The main disease spectrum included tuberculosis, herpes virus infections, pneumocystis jirovecii pneumonia. No matter in the IRIS group or non-IRIS group, the main lymphokines baseline of IL-2, INF-γ reduced and IL-4, IL-10 increased before HAART compared to healthy group (P ＜ 0. 05), which had the tendency to restore balance relations initiating HAART. The lymphokines levels had significant difference between baseline and 6 months initiating HAART (P ＜ 0. 05). The changed levels of lymphokines between IRIS group and non-IRIS group before HAART had significant difference compared to healthy group. IL-2, INF-γ increased level[(11.68 ± 2. 89) pg/ml vs (8.52 ±2.26) pg/ml; (22. 19 ± 6. 22) pg/ml vs (18.34 ±5. 35) pg/ml] and IL-10 decreased level [(19. 21 ± 4. 03) pg/ml vs (23. 19 ± 5.92) pg/ml] had significant difference between IRIS group and non-IRIS group initiating HAART I month(P ＜0. 05). Conclusions The incidence of IRIS during 6 months initiating HAART in HIV/AIDS was 27. 8%, IRIS usually occurred in 1 month initiating HAART. The most common disease spectrum was infectious disease, including tuberculosis and herpes virus infection. Lymphokine of Th1 and Th2 existed unbalance in IRIS group and non-IRIS group before HAART. The unbalance tendency in IRIS group was more obvious. All lymphokines had the trend to recover balance. IL-2, INF-γ significantly increased and IL-10 significantly decreased, which might involve the occurrence of the IRIS.

BACKGROUNDIn tumors the process of apoptosis occurs over an interval of time after chemotherapy. It is important to determine the best time for detecting apoptosis by in vivo imaging. In this study, we evaluated the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using a (99)Tc(m)-labeled Annexin V recombinant with ten consecutive histidines (His10-Annexin V) in a mouse model.

METHODS(99)Tc(m)-His10-Annexin V was prepared by one step direct labeling; radio-chemical purity (RCP) and radio-stability was tested. The binding of (99)Tc(m)-His10-Annexin V to apoptotic cells was validated in vitro using camptothecin-induced Jurkat cells. In vivo bio-distribution was determined in mice by dissection. The human H460 NSCLC tumor cell line (H460) tumor-bearing mice were treated with intravenous paclitaxel 24, 48 and 72 hours later. (99)Tc(m)-His10-Annexin V was injected intravenously, and planar images were acquired at 2, 4 and 6 hours post-injection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and they reflected specific binding of (99)Tc(m)-His10-Annexin V. Mice were sacrificed after imaging. Caspase-3, as the apoptosis detector, was determined by flow cytometry, and DNA fragmentation was analyzed by the terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Nonspecific accumulation of protein was estimated using bovine serum albumin (BSA). The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity.

RESULTS(99)Tc(m)-His10-Annexin V had a RCP > 98% and high stability 2 hours after radio-labeling, and it could bind to apoptotic cells with high affinity. Bio-distribution of (99)Tc(m)-His10-Annexin V showed predominant uptake in kidney, relatively low uptake in myocardium, liver and gastrointestinal tract, and rapid clearance from blood and kidney was observed. The T/NT was significantly increased after paclitaxel treatment, whereas it was low in untreated tumors (T/NT = 1.43 ± 0.18). The %ID/g activity in Group 2 (24 hours), Group 3 (48 hours) and Group 4 (72 hours) after treatment was 2.55 ± 0.73, 3.35 ± 1.10, and 3.4 ± 0.96, respectively. Whereas in the non-treated group, Group 1, %ID/g was 1.10 ± 0.18. The radiotracer uptake was positively correlated to the apoptotic index (r = 0.852, P < 0.01), as well as caspase-3 activity (r = 0.816, P < 0.01).

CONCLUSIONThis study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using (99)Tc(m)- His10-Annexin V.

Animals ; Annexin A5 ; Antineoplastic Agents, Phytogenic ; therapeutic use ; Apoptosis ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Histidine ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Mice ; Organotechnetium Compounds ; Paclitaxel ; therapeutic use ; Radiopharmaceuticals

Objective To study on the effects of Huaweishu granule on cisplatin-induced Beagle dog vomiting models and its mechanism.Methods Cisplatin-induced Beagle dog vomiting models were adopted as research object.The changes of vomiting frequency,latency,serum motilin content in the medulla oblongata and ileum,5-HT and substance P content of these models after using Hua weishu Granule were observed.Results ① Latent period of vomiting of the model group was (60.8±37.1)min; while this period was (137.3± 53.4)min,(122.8 ± 50.7) min,(l16.8±44.6)min,in the Huawei-shu low,medium and high dose group respectively,all showing a statistic difference than the model group (P＜0.05).②Frequency of vomiting in the model group was (270.3±51.8),while this frequence was (111.5±45.0) and (149.5±26.8) in Huawei-shu low and medium dose group respectively; ③ serum motilin in the model group was (0.354±0.098)ng/ml,while serum motilin in Huawei-shu low,medium and high dose group was (0.230±0.074) ng/ml,(0.235± 0.071) ng/ml,and (0.245± 0.062)ng/ml respectively,all lower than the model group (P＜ 0.05).④ Medulla oblongata 5-HT in the model group was (2.028 ±0.198)ng/ml,while medulla oblongata 5-HT in Huawei-shu low,medium and high dose group was (1.620±0.329)ng/ml,(1.194±0.386)ng/ml,and (1.269 ± 0.251) ng/ml respectively,all were lower than the model group (P＜0.05); ileum 5-HT in the model group was (1.634± 0.221)ng/ml,while ileum 5-HT in Huawei-shu low,medium and high dose group was (1.108±0.291)ng/ml,(1.194±0.386)ng/ml,and (1.269 ± 0.251) ng/ml respectively,all were lower than the model group(P＜0.05) ;⑤ ileal substance P content of the model group was (0.356±0.063)ng/ml,while ildeal P content in Huawei-shu low dose group was (0.274±0.064)ng/ml,lower than the model group than(P＜0.05).The medullary substance P content was (0.432±0.021)ng/ml in the model group,while medullary P content in Huawei-shu low,medium and high dose group was (0.370±0.040) ng/ml,(0.385±0.029) ng/ml,and (0.386± 0.041)ng/ml respectively,all were lower than the model group(P＜0.05).Conclusion Huawei-shu granule can prevent cisplatin-induced Beagle vomiting.

OBJECTIVE: To explore the relationship between the expression of EIIIA+ fibronectin in incised wound of rat's skin and injury time. METHODS: The wounding model was established by cutting the dorsal skin of 48 adult SD rats. The rats were sacrificed at the pre-set injury time as immediately, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h. The skin samples were taken at the margin of wound. The expression of the EIIIA? fibronectin was detected by immunohistochemistry and Western blotting and the relationship be- tween its expression and injury time was observed. Results The expression of EIIIA+ fibronectin was not observed immediately. The basal cell of skin began to show positive expression 0.5 h after injury. With the extension of injury time, positive staining became stronger. The value of relative optical density was gradually increased with prolonged injury time by the Western blotting analysis. CONCLUSION The expression of EIIIA+ fibronectin could be used for estimation of injury time in the early stage of skin injury.
Animals ; Fibronectins/metabolism* ; Immunohistochemistry ; Proteins/metabolism* ; Rats ; Rats, Sprague-Dawley ; Skin/metabolism* ; Staining and Labeling ; Time Factors

OBJECTIVETo continuously observe the long-term effects of synovectomy for improving joint damage and quality-of-life in patients with the rheumatoid knee.

METHODSTwenty-one consecutive patients with rheumatoid arthritis (RA) involving 24 knees underwent open synovectomy from November 1988 to January 1997 between November 1988 and January 1997. The changes in radiographic damage were assessed with Larsen score on plain films before and 6 months after surgery with subsequent annual assessment for 8 years, and the functional recovery of the patients was also evaluated with Health Assessment Questionnaire (HAQ) at the same time.

RESULTSThe radiographic joint damage and juxta-articular osteoporosis or bone erosion was ameliorated after surgery in all the patients. Larsen score began to decrease 6 months after the operation, and the best effects were achieved at one year and maintained for at least 5 years after the operation, but then followed by recurrence of joint lesions. HAQ scores were improved after the surgery with the best effects observed 6 months after the operation lasting for over 2 years. HAQ score gradually decreased 4 years after the operation till reaching the preoperative scores.

CONCLUSIONSynovectomy in the patients with rheumatoid knee not only reverses progressive joint damage, but also improves juxta-articular bone erosions and the patients' quality of life. However, radiographic joint damage and functional deterioration may recur due to hyperplasia of the inflammatory synovium in the long term after operation, suggesting that the inflammatory synovium participates in local joint damage with bone erosions and systemic pathologic process of RA.

Adult ; Arthritis, Rheumatoid ; diagnostic imaging ; surgery ; Female ; Follow-Up Studies ; Humans ; Knee Joint ; diagnostic imaging ; surgery ; Male ; Middle Aged ; Radiography ; Synovectomy ; Time Factors

Hydrogel nanoparticles (NPs) were synthesized by precipitation polymerization method with N-isopropyl acrylamide (NIPAm), acrylic acid (Aac), N-tert butyl acrylamide (tBAM) and N,N'-methylene bisacrylamide(Bis) as thermosensitive monomer,negative monomer,hydrophobic monomer and crosslinker, respectively. The morphology of the resulting NPs was characterized by scanning electron microscopy(SEM),and the size and the particle size distribution were investigated by dynamic light scattering (DLS). The dynamics test was also carried out to investigate adsorption property of NPs. The results showed that NPs was spherical with uniform particle size and narrow distribution. NPs had the best adsorption performance to lysozyme when the monomer molar ratio was optimized to Aac 20%,tBAM 40%,NIPAm 38% and Bis 2%. Meanwhile, when the particle size of NPs decreased from 386. 20 nm to 77. 25 nm, the adsorption capacity increased gradually. The adsorption rate could reach up to 67.8% within 5 minutes. The thermosensitive of NPs provided a new candidate for the adsorption and separation of lysozyme with good reusability.

OBJECTIVETo study the expression of CD138 and heparinase in hepatocellular carcinoma (HCC) and its relationship with tumor development, progression, metastasis and recurrence.

METHODSTissue microarray and immunohistochemical study (EnVision method) for CD138 and heparinase was performed on tissue microarray which consisted of 197 cases of HCC, including adjacent non-neoplastic liver tissues, and 66 cases of HCC metastases.

RESULTSThe rates of CD138 expression in HCC and adjacent non-neoplastic liver tissues were 48.7% (96/197) and 65.0% (128/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 61.7% (29/47) and 44.7% (67/150, P < 0.05) respectively. The rate in patients with metastasis was 33.3% (22/66), as compared with 53.6% (45/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 23.3% (7/30) and 61.1% (11/18, P < 0.05) respectively. On the other hand, the rates of expression of heparinase in HCC and adjacent non-neoplastic liver tissues were 35.5% (70/197) and 12.7% (25/197, P < 0.05) respectively. In early-stage and late-stage tumors, the expression rates were 29.8% (14/47) and 37.3% (56/150, P > 0.05) respectively. The rate in patients with metastasis was 48.5% (32/66), as compared with 28.6% (24/84, P < 0.05) in patients without metastasis. In patients with tumor recurrence occurring within or after 1 post-operative year, the expression rates were 50.0% (15/30) and 44.4% (8/18, P > 0.05) respectively. In the 66 cases of metastatic HCC studied, the expression rate of CD138 was lower in the heparinase-positive subgroup (P < 0.05).

CONCLUSIONSLoss of CD138 expression is related to HCC development, progression, metastasis and recurrence. Overexpression of heparinase, when coupled with loss of CD138 expression, may take part in tumor metastasis of HCC.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; metabolism ; secondary ; Female ; Follow-Up Studies ; Heparin Lyase ; metabolism ; Humans ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplastic Cells, Circulating ; metabolism ; Peritoneal Neoplasms ; metabolism ; secondary ; Portal Vein ; Syndecan-1 ; metabolism ; Tissue Array Analysis

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.
Cell Hypoxia ; Cells, Cultured ; Cellular Senescence ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Plasminogen Activator Inhibitor 1 ; genetics ; metabolism ; Serum Response Factor ; genetics ; metabolism ; beta-Galactosidase ; metabolism ; rac1 GTP-Binding Protein ; metabolism

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.
Actins ; biosynthesis ; genetics ; Cytoskeleton ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; Protein-Serine-Threonine Kinases ; metabolism ; Serum Response Factor ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; rho-Associated Kinases ; rhoA GTP-Binding Protein ; physiology

Objective: To explore the effect of ligustrazine on the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes. Methods: Osteoarthritis model was induced by LPS. Chondrocytes were divided into four group: control group, ligustrazine ( 20 μmol/L) group, LPS ( 100 ng/ml) group and ligustrazine ( 20 μmol/L) +LPS ( 100 ng/ml) group. Apoptosis was measured by Hoechst33258 staining. The levels of nitric oxide ( NO), tumor necrosis factor α ( TNF-α) and interleukin ( IL) -6 were detected by ELISA. The protein levels of collagenⅡ, aggrecan, matrix metalloproteinase 13 ( MMP-13), NF-κB P65 and p-NF-κB P65 were tested by Western blot. Results: The LPS-induced abnormal cell morphology and decreased number of cells were ameliorated by ligustrazine ( 20 μmol/L). The apoptosis in LPS group was higher than control group ( P＜0. 05). The LPS-induced enhancive apoptosis was reduced by ligustrazine ( P＜0. 05). Compared with control group, the expression of collagenⅡ and aggrecan was alleviated with increased expression of MMP-13 ( P＜0. 05). The LPS-induced declined expression of collagenⅡ and aggrecan and elevated expression of MMP-13 was inhibited by ligustrazine ( P＜0. 05). The levels of NO, TNF-α and IL-6 in LPS group were higher than control group ( P＜0. 05). The levels of NO, TNF-α and IL-6 in LPS+ligustrazine group were lower than LPS group ( P＜0. 05). Compared with control group, the rate of pP65/P65 in LPPS group was enhanced ( P＜ 0. 05). The LPS-indiced increased rate of p-P65/P65 was decreased by ligustrazine ( P ＜0. 05). Conclusion: Ligustrazine alleviates the LPS-induced apoptosis and inflammatory response of osteoarthritis chondrocytes via inhibiting phosphorylation of NF-κB P65.