Physicochemical properties and cell-based bioactivity of Pu'erh tea polysaccharide conjugates.

Polysaccharide conjugates were prepared from Pu'erh tea and fractionated by DEAE-cellulose DE-52 column chromatography to yield one unexplored polysaccharide-conjugate fraction termed TPC-P with a molecular weight of 251,200Da. DVS (dynamic vapour sorption) result discovered that the humidity condition of long-term preservation for TPC-P is below 70% RH. Although it contained proteins, TPC-P could not bind to the Coomassie Brilliant Blue dyes G250 and R250. The "shoulder-shaped" ultroviolet absorption peak in TPC-P UV-vis scanning spectum ascribe theabrownins that inevitably adsorbed the polysaccharide conjugate. Zeta potential results demonstrated TPC-P aqueous solution merely presented the negative charge properties of polysaccharides instead of acid-base property of its protein section, and had more stability in greater than pH 5.5. No precipitation or haze occurred in the three TPC-P/EGCG aqueous mixtures during their being stored for 12h. The phase separation was observed in aqueous mixtures of TPC-P and type B gelatin. TPC-P possessed the fine stability as a function of temperature heating and cooling between 0 and 55°C. It is proposed that some properties of the covalent binding protein of TPC-P were "shielded" by its polysaccharide chains.

[Correlations of TGF-betaRII, Smad4 and Smad7 expression to clinicopathologic characteristics and prognosis of gastric cancer].

BACKGROUND AND OBJECTIVE: Recent studies have revealed that TGF-beta/Smads signaling pathway plays a pivotal role in the oncogenesis and development of malignant tumors, and may closely relate to the biological behaviors of some malignant tumors, such as gastric carcinoma. This study was to investigate the expression of transforming growth factor-beta receptor II (TGF-betaRII), Smad4 and Smad7 proteins in gastric carcinoma, and explore their correlations to clinicopathologic characteristics and prognosis of gastric carcinoma. METHODS: The expression of TGF-betaRII, Smad4, and Smad7 was detected by SABC immunohistochemistry and tissue microarray which containing 200 specimens of primary human gastric carcinoma and 56 specimens of adjacent gastric tissue. RESULTS: The positive rates of TGF-betaRII, Smad4, and Smad7 in gastric carcinomas were 25.5%, 67.0%, and 47.0%, respectively. TGF-betaRII expression was related with depth of invasion, lymph node metastasis, tumor differentiation, and Lauren classification (Chi2=6.214, Chi2=11.308, Chi2=14.633, and Chi2=8.216, respectively, all P<0.05). Smad4 expression was related with tumor differentiation and Lauren classification (Chi2=16.162 and Chi2=13.100, all P<0.05). Smad7 expression was related with tumor differentiation and Lauren classification (Chi2=4.710 and Chi2=5.297, all P<0.05). Smad4 expression was positively correlated to TGF-betaRIIexpression (r =0.191, P=0.007). Smad4 expression was related with patients' survival (Chi2=4.090, P=0.043). CONCLUSIONS: Abnormal expression of TGF-Smad signaling pathway proteins occurs in gastric carcinoma. Smad4-positive gastric carcinoma patients have better prognosis than Smad4-negative patients.

[Epidemiological study on status of Helicobacter pylori in children and teenagers in Wuwei city, Gansu province].

OBJECTIVE: To investigate the prevalence of Helicobacter pylori (Hp) infection among children aged 3 to 18 years old of Wuwei city, Gansu province. METHODS: The study was based upon a personal questionnaire and a determination of Hp antigen using the Hp stool antigen test (HpSA) method. A total of 938 subjects and 96 families were selected in Wuwei city. Eighty children and teenagers with a definite positive Hp stool antigen test were examined by serum Western blotting method. RESULTS: The prevalence of Hp was 72.3% (678/938) with no age difference. Prevalence of Hp infection was correlated with type of dwelling, occupation of parents, drinking water source, kindergarten attendance, consumption of raw vegetables, a poor oral hygiene and breast feeding etc. According to the multivariate analysis, drinking water source, kindergarten attendance and consumption of raw vegetables were most strongly associated with prevalence of Hp in children and adolescents. The infection rate of parents whose children were infected with Hp was higher than that of those whose children were not infected [82.3% (121/147) vs 47.4% (18/38), chi(2) = 19.736, P < 0.05]. The antibody responses of 57 samples (71.3%) from 80 children were of type I Hp and 23 samples (28.7%) type II. CONCLUSIONS: Hp infective rate is high in Wuwei city. The data support maternal-child and sibling-sibling transmission as the primary transmission routes of Hp. The results of serological analysis confirm that the majority of Wuwei Hp infection is of type I.

[Expression of cyclooxygenase-2 and urokinase plasminogen activator in gastric carcinoma and the clinical significance thereof].

OBJECTIVE: To investigate expression of cyclooxygenase-2 (COX-2) and urokinase plasminogen activator (uPA) in gastric carcinoma and the clinical significance thereof. METHODS: Strepavidin-peroxidase method was used to detect the expression of COX-2 and uPA in 192 specimens of gastric carcinoma, 56 specimens of paracancer tissues obtained during operation. Immunohistochemical double staining was used to detect the microvessel density (MVD) and microlymphatic density (MLD). Thirty specimens of normal gastric mucosa obtained during gastroscopy were used as controls. RESULTS: The positive rates of COX-2 in the gastric carcinoma and paracancer tissues were 67.7% and 62.5% respectively, both significantly higher than that of the control group (40.0%, both P < 0.05). The positive expression of COX-2 in gastric carcinoma was significantly related with the depth of invasion and MVD (both P < 0.05). The positive rates of uPA in the gastric carcinoma, paracancer tissue were 78.1% and 44.6% respectively, both significantly higher than that of the control tissues (6.7%, both P < 0.01) and there was a significant difference in the positive rates of uPA between the first 2 groups too (P < 0.01). The positive expression of uPA in gastric carcinoma was significantly related with lymph node metastasis, depth of invasion, Lauren typing, differentiation, MVD, and MLD (P < 0.05, P < 0.01). COX-2 expression was positively linked with uPA expression (r = 0.167, P = 0.021). The survival time of the uPA positive group was (38 +/- 4) months, significantly shorter than that of the negative group [(54 +/- 6) months, P < 0.05]. The survival time of the group positive in both COX-2 and uPA was (27 +/- 3) months, significantly shorter than that of the single positive or double negative groups [both (58 +/- 4) months, both P < 0.01). CONCLUSION: COX-2 and uPA are highly expressed in gastric carcinoma. COX-2 expression is positively linked with uPA expression. COX-2 and uPA in the gastric carcinoma participate in the development of gastric cancer in the early process. uPA is significantly related with the survival time.

Both innate immunity and type 1 humoral immunity to Streptococcus pneumoniae are mediated by MyD88 but differ in their relative levels of dependence on toll-like receptor 2.

Little is known regarding the role of Toll-like receptors (TLRs) in regulating protein- and polysaccharide-specific immunoglobulin (Ig) isotype production in response to an in vivo challenge with an extracellular bacterium. In this report we demonstrate that MyD88(-/-), but not TLR2(-/-), mice are markedly defective in their induction of multiple splenic proinflammatory cytokine- and chemokine-specific mRNAs after intraperitoneal (i.p.) challenge with heat-killed Streptococcus pneumoniae capsular type 14 (S. pneumoniae type 14). This is correlated with analogous responses in splenic cytokine protein release in vitro following addition of S. pneumoniae type 14. Consistent with these data, naive MyD88(-/-), but not TLR2(-/-), mice are more sensitive to killing following i.p. challenge with live S. pneumoniae type 14, relative to responses in wild-type mice. However, prior immunization of MyD88(-/-) mice with heat-killed S. pneumoniae type 14 protects against an otherwise-lethal challenge with live S. pneumoniae type 14. Surprisingly, both MyD88(-/-) and TLR2(-/-) mice exhibit striking and equivalent defects in elicitation of type 1 IgG isotypes (IgG3, IgG2b, and IgG2a), but not the type 2 IgG isotype, IgG1, specific for several protein and polysaccharide antigens, in response to i.p. challenge with heat-killed S. pneumoniae type 14. Of note, the type 1 IgG isotype titers specific for pneumococcal surface protein A are reduced in MyD88(-/-) mice but not TLR2(-/-) mice. These data suggest that distinct TLRs may differentially regulate innate versus adaptive humoral immunity to intact S. pneumoniae and are the first to implicate a role for TLR2 in shaping an in vivo type 1 IgG humoral immune response to a gram-positive extracellular bacterium.

4-1BB (CD137) differentially regulates murine in vivo protein- and polysaccharide-specific immunoglobulin isotype responses to Streptococcus pneumoniae.

4-1BB (CD137) is induced on activated CD4(+) and CD8(+) T cells and delivers a costimulatory signal upon binding the 4-1BB ligand (4-1BBL) expressed on antigen-presenting cells. Induction of 4-1BB is dependent on activation via the T-cell receptor (TCR) and possibly CD28. It was previously demonstrated that both an in vivo protein (pneumococcal surface protein A [PspA])- and polysaccharide (phosphorylcholine [PC] determinant of teichoic acid)-specific immunoglobulin (Ig) isotype response to Streptococcus pneumoniae was dependent on CD4(+) TCRalphabeta(+) T cells and B7-dependent costimulation through CD28. We thus postulated that 4-1BB costimulation would also play a role in regulating the in vivo anti-PspA and anti-PC response to S. pneumoniae. We demonstrate that mice genetically deficient in 4-1BBL elicit a markedly reduced IgM and IgG anti-PC but normal primary and secondary IgG anti-PspA responses to S. pneumoniae relative to those for wild-type mice. However, injection of an agonistic anti-4-1BB monoclonal antibody (MAb), while having no significant effect on the anti-PC response, strongly inhibits the primary anti-PspA response, the generation of PspA-specific memory, and germinal center formation but does not induce a lasting state of tolerance. In contrast, anti-4-1BB MAb has no effect on the anti-PspA response when injected only at the time of secondary immunization. Delay of the addition of anti-4-1BB leads to progressively less inhibition of the primary response up to day 8. This inhibition is independent of CD8(+) T cells and is associated with the expansion of CD4(+) T cells with an activated phenotype, which is partly dependent on B7-dependent costimulation. These data are the first to suggest a stimulatory role for endogenous 4-1BB-4-1BBL interactions during a humoral immune response to a pathogen and further underscore significant differences in costimulation requirements for an in vivo protein- versus polysaccharide-specific Ig isotype response to an extracellular bacterium.

The mechanism underlying T cell help for induction of an antigen-specific in vivo humoral immune response to intact Streptococcus pneumoniae is dependent on the type of antigen.

Little is known concerning the role of T cells in regulating an anti-polysaccharide Ig response to an intact pathogen. We previously reported that the in vivo Ig responses to Streptococcus pneumoniae (strain R36A), specific for pneumococcal surface protein A (PspA) and for the phosphorylcholine (PC) determinant of C-polysaccharide, were both dependent on TCR-alphabeta(+) T cells and B7-dependent costimulation, although only PspA-specific memory was generated. In this report, we show that the T cell help underlying these two Ag-specific Ig responses is distinct. Using H-Y-specific T cell transgenic mice made "nonleaky" by crossing with mice genetically deficient for TCR-alpha, we demonstrate that the T cell help for the anti-PC, in contrast to the anti-PspA, response is TCR-nonspecific and occurs normally in the absence of germinal center formation, although it is still dependent on B7-dependent costimulation. Consistent with these data, we demonstrate, using cathepsin S(-/-) mice, that although the anti-PC response is largely dependent on CD4(+) T cells, there is a reduced (or lack of) dependence, relative to the anti-PspA response, on the generation of new peptide-MHC class II complexes. In this regard, the T cell help for an optimal anti-PC response is delivered more rapidly than that required for an optimal anti-PspA response. Collectively, these data demonstrate a novel accelerated TCR-nonspecific B7-dependent form of T cell help for augmenting a polysaccharide-specific Ig response to an intact bacterium without the generation of memory.

Endogenous pro- and anti-inflammatory cytokines differentially regulate an in vivo humoral response to Streptococcus pneumoniae.

Proinflammatory cytokines play a critical role in innate host defense against extracellular bacteria. However, little is known regarding the effects of these cytokines on the adaptive humoral response. Mice injected with a neutralizing anti-tumor necrosis factor alpha (TNF-alpha) monoclonal antibody (MAb) at the time of primary immunization with intact Streptococcus pneumoniae (strain R36A) showed a substantial reduction in both the primary immunoglobulin G (IgG) response specific for the cell wall protein, pneumococcal surface protein A (PspA), as well as in the development of PspA-specific memory. In contrast, anti-TNF-alpha MAb injected only at the time of secondary immunization with R36A failed to alter the boosted anti-PspA response. TNF-alpha was required only within the first 48 to 72 h after primary immunization with R36A and was induced both by non-B and non-T cells and by lymphoid cells, within 2 to 6 h after immunization, with levels returning to normal by 24 h. Thus, the early innate release of TNF-alpha was critical for optimal stimulation of the subsequent adaptive humoral response to R36A. Additional proinflammatory (interleukin 1 [IL-1], IL-6, IL-12, and gamma interferon [IFN-gamma]) as well as anti-inflammatory (IL-4 and IL-10) cytokines were also transiently induced. Mice genetically deficient in IL-6, IFN-gamma, or IL-12 also showed a reduced IgG anti-PspA response of all IgG isotypes. In contrast, IL-4(-/-) and IL-10(-/-) mice immunized with R36A showed a significant elevation in the IgG anti-PspA response, except that there was decreased IgG1 in IL-4(-/-) mice. In this regard, a marked enhancement in the induction of proinflammatory cytokines was observed in the absence of IL-10, relative to controls. Ig isotype titers specific for the phosphorycholine determinant of C-polysaccharide were similarly regulated, but to a much more modest degree. These data suggest that proinflammatory and anti-inflammatory cytokines differentially regulate an in vivo protein- and polysaccharide-specific Ig response to an extracellular bacteria.