[The progress and challenges in immunotherapy for the difficulty- to- treat liver diseases].

As a digestive organ, the liver has the functions of metabolism, synthesis, and detoxification. It is also an immune organ and plays an important role in maintaining anti-infection, autoimmune stability, and anti-tumor. In particular, the liver has unique immunological advantages. Its immune cells can maintain the liver's immune homeostasis and participate in immunoregulation. A variety of immunotherapy is used in clinical trials for the treatment of difficult and critical liver diseases. This review mainly summarizes the recent clinical trials of immunotherapy in chronic hepatitis B, cirrhosis, hepatocellular carcinoma, and autoimmune liver disease.

[Prenatal lead exposure related to cord blood brain derived neurotrophic factor (BDNF) levels and impaired neonatal neurobehavioral development].

OBJECTIVE: To explore the relationship between umbilical cord blood brain-derived neurotrophic factor (BDNF) and neonatal neurobehavioral development in lead exposure infants. METHODS: All infants and their mother were randomly selected during 2011 to 2012, subjects were selected according to the umbilical cord blood lead concentrations, which contcentration of lead was higher than 0.48 µmol/L were taken into high lead exposure group, about 60 subjects included. Comparing to the high lead exposure group, according to gender, weight, pregnant week, length and head circumferenece, the level of cord blood lead concentration under 0.48 µmol/L were taken into control group, 60 cases included. Lead content was determined by graphite furnace atomic absorption spectrometry. Neonatal behavioral neurological assessment (NBNA) was used to determine the development of neonatal neuronal behavior. The content of BDNF was detected by ELISA. Comparing the BDNF and the NBNA score between two groups, and linear correlation was given on analysis the correlation between lead concentration in cord blood and BDNF, BDNF and the NBNA score. RESULTS: Lead content in high exposure group was (0.613±0.139) µmol/L, and higher than (0.336±0.142) µmol/L in low exposure group (t=3.21, P<0.001) . NBNA summary score (36.35±1.86), active muscle tension score (6.90±0.27) and general assessment score (5.93±0.32) in high exposure group were lower than those (38.13±0.96, 7.79±0.35, 6.00±0.00) in low exposure group (t values were 8.21, 10.23, 2.32, respectively, P values were <0.001, <0.001 and 0.037) . BDNF content in high exposure group which was (3.538±1.203) ng/ml was higher than low exposure group (2.464±0.918) ng/ml (t=7.60, P<0.001). The correlation analysis found that the cord blood BDNF content was negatively correlated with NBNA summary score, passive muscle tension and active muscle tone score (r was -0.27, -0.29, -0.30, respectively, P values were <0.001, respectively) . CONCLUSION: Prenatal lead exposure results poor neonatal neurobehavioral development and cord blood BDNF was negatively correlated with neonatal neurodevelopment, may serve as a useful biomarker.

Aging affects the regeneration of the CD8+ T cell compartment in bone marrow transplanted mice.

A chimeric mouse model has been used to determine the effect of aging on the differentiation of CD8+ T cells and on the regeneration capacity of the mature peripheral T cell pool after radiation induced depletion. Bone marrow cells from Thy 1.1+ mice were transplanted into lethally irradiated young or aged mice (Thy 1.2+). After 6 weeks, splenic CD8+ T cells were subjected to phenotypic and functional examinations by flow cytometry. Both young and aged mice were able to develop donor derived (Thy 1.1+) CD8+ T cells. Although the absolute number of T cells was reduced in aged recipients, the ratio of CD4+ to CD8+ T cells of donor-origin was the same in young Thy 1.1+ control mice as it was in both young and aged chimeric mice, indicating that aging has no effect on the ratio of CD4+ to CD8+ T cells produced by the thymus. However, the percentage of CD8+ cells in the total Thy 1.2+ (host-origin) T cell population was significantly higher in young chimeric mice than in age-matched Thy 1.2+ control mice (P < 0.01), suggesting that a significant over expansion of the Thy 1.2+ CD8+ subset occurred in young mice during regeneration. The Thy 1.1+ CD8+ T cells that developed in young hosts were of a naive phenotype with a majority of cells expressing a low level of CD44. In contrast, the majority of those that developed in the aged host displayed a memory phenotype with a high percentage of cells being CD44hi. In addition, the production of IL-4 and IFN-gamma by Thy 1.1+ CD8+ T cells was affected by the age of the host. A greater fraction of aged Thy 1.1+ CD8+ T cells could be induced to produce either IFN-gamma or IL-4 than young CD8+ T cells. These results suggested that the aged microenvironment has a significant effect on newly developed CD8+ T cells and that the age of the microenvironment also influences the regeneration capacity of CD8+ T cells.

Activation of human peripheral blood T cells does not lead to increased P-glycoprotein expression.

The expression of P-glycoprotein (Pgp) on normal human lymphocytes, and its drug exclusion capacity, implies that Pgp might be involved in cytokine secretion. We used two-color flow cytometry to detect simultaneously Pgp expression and IL-2 accumulation in resting and mitogen-activated human lymphocytes. Among resting lymphocytes from five healthy donors less than 1% were Pgp+ as determined by reactivity with the anti-Pgp monoclonal antibody (mAb) 4E3. The percentage of Pgp+ lymphocytes increased to 3% after 24 hr of mitogenic stimulation that induced maximal production of cytoplasmic IL-2. The percentage of lymphocytes that coexpressed membrane Pgp and cytoplasmic IL-2 accounted for < 10% of the total IL-2 producing lymphocytes. Finally, mitogen-induced cytoplasmic IL-2 accumulation was enhanced by stimulation in the presence of monensin but not the Pgp functional inhibitor verapamil. Because mAb 4E3 detected lower than expected numbers of Pgp+ lymphocytes, we compared the binding of mAbs MRK16 and 4E3 concomitant with doxorubicin (DOX)-uptake by K562 and R7 tumor cells and purified CD8+ lymphocytes. The MRK16 mAb was found to be sensitive but not very specific (30%). In contrast, the sensitivity of 4E3 was equivalent to MRK16 (98%) and was highly specific (98.5%). There was also a positive association between DOX efflux and the level of Pgp expression as detected by 4E3 but not MRK16. Thus, human T cells do not markedly up-regulate their expression of functional Pgp molecules as detected by mAb 4E3 following activation, suggesting that Pgp does not play a major role in IL-2 secretion by activated T cells.

Characterization of peroxidase:anti-peroxidase immune complexes by capillary zone electrophoresis and high-performance size-exclusion chromatography.

Determination of the molecular constituents of commercial peroxidase:anti-peroxidase (PAP) preparations is necessary for the proper interpretation of PAP applications based on competitive binding assay. Capillary zone electrophoresis with field 300 V/cm, 40 cm capillary length (20 cm effective length), and high-performance size exclusion chromatography equipped with Superose 12 HR10/30 column revealed that a PAP preparation used for Fc gamma receptor studies contained multiple sizes of immune complexes, an excess amount of free peroxidase, and little or no free anti-peroxidase antibody. The antibody:antigen ratios of the three major immune complex components were 2:2, 1:2, and 1:1. These techniques provide useful methods of qualitative, as well as quantitative analysis of PAP preparations.

The age-dependent cytokine production by murine CD8+ T cells as determined by four-color flow cytometry analysis.

Flow cytometry has been used to simultaneously examine intracellular cytokine production and expression of CD44 and CD45RB by murine CD8+ T cells derived from young (2-3 months) or aged (18-22 months) mice. Cytokine production by aged CD8+ T cells differs from that by CD8+ T cells derived from young animals in that a significantly higher percentage of the aged can be triggered to produce interleukin (IL)-4, interferon (IFN)-gamma, and tumor necrosis factor alpha (TNF alpha). Conversely, a greater fraction of young CD8+ T cells produce IL-2. Aged mice not only have a higher percentage of CD8+/CD44hi T cells, but also a larger fraction of these cells are IFN-gamma+ and IL-4+, while a lower fraction are IL-2+ than is observed in young CD8+/CD44hi T cells. In terms of relative contribution to total cytokine synthesis, a greater fraction of CD8+ T cells produce IFN-gamma and IL-4 than in CD4+ T cells, whereas CD4+ T cells are the major producers of IL-2.

L-Canavanine modulates cellular growth, chemosensitivity and P-glycoprotein substrate accumulation in cultured human tumor cell lines.

L-Canavanine (L-CAV) is a naturally occurring L-arginine analog that induces the formation of non-functional proteins in a variety of organisms. Previous studies have shown that L-CAV is cytotoxic for several human tumor cell lines. In this study, we have evaluated the cytotoxicity of L-CAV for both parental and multi-drug resistant (MDR) human tumor cells. We have also determined the effect of L-CAV exposure on cellular expression and activity of the MDR P-glycoprotein (P-gp) membrane efflux pump, and the effect of L-CAV on cellular accumulation of P-gp substrates. The effect of pre-treatment with non-cytotoxic doses of L-CAV on cellular sensitivity to ten standard antineoplastic agents was also evaluated, in order to assess the chemosensitization potential of L-CAV. 3-(4,5-Dimethylthiazol-)2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assays revealed that the MDR variants of human uterine sarcoma and leukemic cells were equally sensitive to L-CAV as compared with their respective parental controls. Although the presence of free L-CAV in the uptake media did not influence cellular accumulation of P-gp substrates, cells cultured for 72 h in 250 microM L-CAV accumulated from 16 to 23% less P-gp substrate than untreated controls. Although L-CAV-cultured sarcoma cells accumulated 17% less doxorubicin (DOX) than untreated controls, they were three times more sensitive to its cytotoxic effects. L-CAV-treated cells were also significantly more sensitive to cisplatin, 5-fluorouracil, mitoxantrone and bleomycin than were untreated controls. Indirect immunofluorescence revealed that 72-h exposure to as much as 1000 microM L-CAV did not alter cellular expression of P-gp. These studies suggest that L-CAV may be equally cytotoxic for both parental and MDR tumor cells, and that L-CAV neither induces the expression of, nor is a substrate for, P-gp. The observation that L-CAV pre-treatment reduces cellular accumulation of DOX, yet sensitizes tumor cells to DOX and other DNA-targeting antineoplastic drugs, suggests a role for L-CAV as a chemosensitizer for the chemotherapy of cancer.

Use of peroxidase:anti-peroxidase immune complexes as markers for Fc gamma receptors in vitro.

In species for which monoclonal antibodies are not yet available, the demonstration of Fc receptors has relied on a variety of ligand-based assays including the binding of antibody-coated erythrocytes, radiolabeled monomeric immunoglobulin, and non-physiologic aggregates of immunoglobulin. In order to study the binding of small immune complexes to Fc receptors on canine monocytes, a new method was developed using an enzyme-linked immune complex. The ability of rabbit polyclonal peroxidase:anti-peroxidase (PAP) immune complexes to bind to freshly isolated canine peripheral blood monocytes was characterized using standard ELISA techniques. The binding of rabbit polyclonal PAP to monocytes was time and concentration dependent and reversible. This binding was saturable with increasing concentrations of PAP and could be blocked by soluble rabbit IgG or rabbit Fc fragments. The blocking and saturation curves for canine monocytes were suggestive of multiple classes of Fc gamma binding sites. In contrast to intact PAP complexes, the binding of F(ab) PAP preparations or free horseradish peroxidase was minimal. The use of commercially available PAP preparations provides a reproducible, inexpensive, and non-radioactive measure of Fc gamma receptor binding on canine cells. In addition, these findings suggest caution in using heterologous PAP as a histochemical reagent in tissues expressing Fc gamma receptors.

[Influence of colchicine upon morphology of intervertebral disc of rabbit].

The morphological changes of intervertebral disc of rabbit in the culture liquids of containing colchicine was observed by microscopy and transmission microscopy and hydroxyproline density of culture liquids was measured by stages. Meanwhile, set up control groups. Undiscovery that colchicine can produce discolysis and shrinking. We regard as major mechanism of colchicine treating herniated of intervertebral disc is anti-inflammation and anti-microtubule. We advance opposite viewpoint for Michael R. Rask's hypothesis which colchicine can produce herniated of intervertebral disc shrinking.