Active oxygen species generated by monocytes and polymorphonuclear cells in Crohn's disease

Chemiluminescence (CL) analysis of monocytes and polymorphonuclear cells (PMNs) was performed on 13 patients with Crohn's disease (CD) and 10 healthy volunteers. The percentages of monocyte populations in mononuclear cells obtained from the patients with CD were greater than those from the healthy volunteers, but the numbers of PMNs were not different between the two groups. The peak level of phorbol myristate acetate (PMA)-induced CL activity generated by diluted whole blood from the patients with CD was more significantly elevated than that from the healthy volunteers, whereas the peak levels of opsonized zymosan-induced CL activity did not differ between the two groups. In monocytes, the peak levels of both PMA- and opsonized zymosan-induced CL activity were significantly higher in the patients with CD than in the healthy volunteers. CL in PMNs, however, showed no significant difference between CD and controls. It is suggested that monocytes of CD have a large capacity to generate active oxygen species. The present study suggests that excessive active oxygen species released by monocytes and perhaps macrophages may play an important role in formation of the intestinal lesions in CD.This work was supported by the Grant of Tokuteishitsukan from the Japanese Ministry of Welfare and Health.     

Toxicodynamics of tumour promoters of mouse skin

Summary In binding competition assays using a protein kinase C preparation from mouse brain (particulate fraction)³H-labelled 12-O-tetradecanoylphorbol-13-acetate (TPA), for a series of new diterpene esters (DTE) the relative binding affinity [rba=K _i ^a (TPA)/K _i ^a (DTE)] in relation to TPA was determined. A wide range of values was noticed, some of the DTE binding more strongly than TPA (rba >1), others binding less strongly than TPA (rba <1) In comparative terms, competition for specific binding sites appears to correlate better with irritant than with promoting acitvity of the DTE. Using mouse peritoneal neutrophils, binding of [³H]-TPA was determined by a modification of the cold-acetone filter assay; saturation of high-affinity sites (K _d ^a =0.2 nM) was obtained at concentrations 1 nM, but there was also evidence for specific binding at low-affinity sites (K _d ^a =26 nM). Induction of chemoluminescence in the presence of luminol in mouse peritoneal neutrophils with a set of DTE usually elecited two peaks; at concentrations 10 nM DTE a short-lived, spike-like response lasting only from 0 to about 5 min (phase A) ist followed by a plateau response from about 5–120 min (phase B). This latter phase of chemoluminescence stimulation with luminol correlated well with theirritant potential of the DTE used. The sequence of the two phases can be inverted partially by using first TPA at 2,5 nM followed by a quick concentration increase to 100 nM; this indicates two different concentration-dependent events. As regards the intensity of the chemoluminescent response, quantitative but not qualitative differences between DTE were observed, which show some correlation with strong and weak tumour-promoting activity. Inhibition studies suggest the involvement of the myeloperoxidase/H₂O₂/Cl^– system in the luminogenic response; it is suggested that the release of hypochlorite or a closely related oxidant may be instrumental in tumour promotion.Abbreviations DMSO dimethylsulphoxide - DTE diterpene ester - K10 Gnidia factor K10 (Kraussianin) - K _i ^a ,K _d ^a apparent inhibition and dissociation constants - muPMN mouse peritoneal neutrophils - P2 Pimelea factor P2 - PDD phorbol-12,13-didecanoate - RPA 12-O-retinoylphorbol-13-acetate - rba relative binding affinity - TPA 12-O-tetradecanoylphorbol-13-acetate - 3-TI 3-O-tetradecanoylingenol See loc. cit. Kloz et al. 1989 for I. Comm. of this series     

Comparison of the sensitivity of NAT using pooled donor samples for HBV and that of a serologic HBsAg assay

BACKGROUND: Studies were conducted using samples from early and late-stage HBV-infected persons to determine the pool size at which PCR had better sensitivity than a sensitive HBsAg chemoluminescence immunoassay (CLIA-HBsAg). STUDY DESIGN AND METHODS: HBV seroconversion panels were tested for HBsAg by CLIA and for HBV DNA by nested PCR (95% hit rate: 100 copies/mL); PCR was carried out at various dilutions. HBV serologically positive samples that were detected from the simultaneous screening of 540,161 routine whole-blood donations using CLIA-HBsAg and agglutination assays were also characterized for additional markers of HBV infection. RESULTS: In 9 of 10 HBV seroconversion panels, PCR had better sensitivity than CLIA-HBsAg at dilutions of 1-in-25 or lower. Of 65 CLIA-only confirmed-positive donor samples (agglutination assay-negative), 8 represented early infection, 2 of which were PCR positive at a 1-in-50 dilution but negative at a 1-in-100 dilution. Only 2 of 47 samples from probable late-stage HBV infection that were positive on CLIA only were PCR positive with 0.1-mL sample volume and the S-region primer; the remaining 45 samples required a 1.0-mL sample input and C-region primer for increased PCR positivity. The remaining 10 CLIA-only confirmed-positive donor samples were from HBV vaccine recipients. None of the 12 CLIA- and HBsAg-negative donor samples that were strongly anti-HBc reactive could be detected by PCR at any dilution; all 12 were PCR positive when undiluted, but 4 required a 1.0-mL input volume for PCR positivity. CONCLUSION: For the detection of samples representing early-stage HBV infection, PCR at dilutions of 1-in-25 or lower (equivalent to a pool of < or =25 members) had greater sensitivity than CLIA-HBsAg. In contrast, samples from late-stage HBV infection were detected by PCR only with undiluted samples (0.1-mL or 1.0-mL input volumes), regardless of CLIA-HBsAg reactivity. Therefore, although NAT using minipools of 25 donations or less may be effective for the detection of early-stage HBV infection, it may not be effective for the detection of persistent HBV infection.     

化学发光法检测慢性乙型肝炎肝纤维化四项标志结果分析

目的探讨应用化学发光法定量检测肝纤维化指标透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原氮端肽(PⅢNP)、Ⅳ型胶原(CⅣ)对肝纤维化的诊断价值。方法应用化学发光法检测了196例乙型肝炎病毒感染者血清样本的肝纤维化指标。其中乙型肝炎病毒携带者50例,慢性乙型肝炎患者48例,慢性重肝36例,乙型肝炎肝硬化患者62例,正常对照40例,并对结果进行统计学分析。结果肝纤维化四项指标(HA、LN、PⅢNP、CⅣ)在乙型肝炎携带者、慢性乙型肝炎、慢性重肝、乙型肝炎肝硬化测定值之间差异有统计学意义(P<0.05)。测定值升高的幅度为:慢性重肝>乙型肝炎肝硬化>慢性乙型肝炎>乙型肝炎携带者。结论应用化学发光法动态检测肝纤维化四项指标是指导临床判断肝纤维化程度的非损伤性的良好检测方法。     

Chemoluminescence generation and MTT dye reduction by polymorphonuclear leukocytes from periodontal disease patients

Alterations of polymorphonuclear leukocytes (PMNs) functions have been reported in patients with severe forms of some periodontal disease. In this study we evaluated the chemoluminescence generation and MTT dye reduction by human PMN in patients with juvenile periodontitis (JP), rapidly progressive periodontitis (RPP) and adult periodontitis (AP) during protein kinase C (PKC) activation or during the phagocytosis of opsonized zymosan. The results demonstrated that only PMNs of JP patients showed a decreased chemoluminescence generation and MTT dye reduction during the phagocytosis of opsonized zymosan (p < 0.05). The time to reach the maximal peak during the PKC activation on the chemoluminescence reaction was evaluated and JP PMNs patients demonstrated a depressed value (7.0 ± 0.4 min) compared with healthy volunteers (13.8 ± 0.5 min). The etiology and importance of such cellular alterations in the immunopathogenesis of the periodontal disease are discussed.     

IFN alpha-2a protects mice against a helminth infection of the liver and modulates immune responses

BACKGROUND & AIMS: Hepatic alveolar echinococcosis (AE), caused by the larval growth of Echinococcus multilocularis, is one of the most lethal helminthic diseases with no satisfactory treatment. Advances in the understanding of the host's immune response (Th2 responses associated with a progressive form of AE), have driven the research towards immune stimulation as an alternative possibility to treat patients. We previously reported clinical stabilization associated with a shift from a Th2 to a Th1 cytokine profile in an AE patient treated with interferon (IFN)alpha. METHODS: The effects of recombinant IFN alpha-2a were analyzed in the susceptible C57BL/6J E. multilocularis infected mice. Parasitic burden, macrophage functions, and specific T-cell responses were studied 15, 45, and 90 days postinfection. RESULTS: After 90 days postinfection, 75% of infected IFN alpha-2a-treated mice had no hepatic lesions and half were fully protected. IFN alpha-2a treatment markedly decreased the abnormally elevated production of IL-10 in both spleen cell cultures and peritoneal macrophage cultures from infected mice and restored phagocytosis and oxidative metabolism of macrophages. It also inhibited IL-6 and IL-13 antigen-induced secretions in spleen cell cultures. CONCLUSIONS: Through its immunoregulatory properties, IFN alpha-2a may be effective in a helminthic liver infection and is a promising candidate for clinical application in AE.     

Effect of Lysosomotropic Form of Amphotericin B on Functional State of Phagocytes in Experimental Candidiasis

Effect of antifungal preparation amphotericin B and its lysosomotropic composition with dialdehyde-dextran on functional state of phagocytizing cells in the dynamics of granulomatous inflammation induced by C. albicans was studied on CBA mice. A stimulating effect of amphotericin B on the production of reactive oxygen species by peritoneal and bone marrow phagocytes was observed, while lysosomotropic form of the antibiotic did not stimulate generation of oxygen radicals. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Suppl. 1, pp. 13–15, 2008     

Expression patterns of claudin-5 and its related signals during luteal regression in pseudopregnant rats: The enhanced effect of additional PGF treatment

To study the expression patterns of claudin-5 and its related signals during luteal regression in rats, a sequential PMSG/hCG treatment paradigm was used to obtain a single, well-defined generation of corpus luteum (CL). A total of 35 rats were treated with one PGF or two PGF at an interval of 24?h from day 7 of pseudopregnancy to induce CL regression. Serum and ovaries were collected at 0, 2, 4, 8 or 24?h after one PGF injection (1 PGF), 2 or 24?h after two PGF injections (2 PGF). The serum progesterone level was detected by RIA; the ovarian expression of claudin-5, the phosphorylations of STAT3 (p-STAT3), Akt (p-Akt), ERK1/2 (p-ERK) and p38 MAPK (p-p38) were detected by western blot, real-time PCR and IHC. Results showed that serum progesterone (P₄) decreased after PGF treatment. Claudin-5 mRNA decreased at 4?h and 8?h after 1 PGF and 2?h after 2 PGF, and claudin-5 protein decreased at 4?h after 1 PGF. p-STAT3 increased at 4?h after 1 PGF and 2?h after 2 PGF. p-ERK increased at 2?h after 2 PGF. The level of p-Akt decreased at 4?h after 1 PGF. PGF treatment did not alter the phosphorylation of p38 MAPK at any time points in this study. IHC results revealed that claudin-5 was expressed in the nuclei and cytoplasm of steroidogenic cells and in the vessels, while PGF induced-p-STAT3 was expressed uniformly in the cytoplasm of luteal steroidogenic cells. In conclusion, PGF treatment decreased the expression of claudin-5 and the additional PGF treatment enhanced the decrease in claudin-5 mRNA expression and the increases in ERK1/2 and STAT3 phosphorylation in the corpus luteum of pseudopregnant rats, which will contribute new information to the further study of molecular mechanism of luteal regression.     

化学发光法检测慢性乙型肝炎病毒感染者肝纤维化四项指标结果分析

目的:探讨应用化学发光法定量检测肝纤维化指标透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原氮端肽(PⅢNP)、Ⅳ型胶原(C-Ⅳ)对肝纤维化的诊断价值.方法:应用化学发光法检测196例慢性乙型肝炎病毒(HBV)感染者血清肝纤维化指标,其中HBV携带者50例,慢性乙型肝炎(CHB) 48例,慢性重型肝炎(简称慢重肝)36例,乙型肝炎肝硬化62例,正常对照40例,并对结果进行统计学分析.结果:患者HA、LN、PⅢNP、C-Ⅳ在HBV携带者、CHB、慢重肝、乙型肝炎肝硬化患者之间比较,差异有显著性意义(P＜0.05).测定值升高的顺序为:慢重肝＞乙型肝炎肝硬化＞ CHB＞ HBV携带者.结论:应用化学发光法动态检测肝纤维化四项指标是指导临床判断肝纤维化程度的非损伤性的良好检测方法.