Use of the enzyme-linked immunosorbent assay for the early diagnosis ofMycoplasma pneumoniae infection

IgM and IgG antibodies toMycoplasma pneumoniae were measured in 147 sera from four groups of patients by means of an indirect enzyme-linked immunosorbent assay (ELISA) and the results compared with those obtained by other methods. A good correlation was demonstrated between the complement fixation test and ELISA-IgM and to a lesser extent ELISA-IgG; for the metabolic inhibition test the reverse was the case. The indirect haemagglutination test appeared to detect mainly IgM antibodies. Low levels of IgM antibodies were dectected by ELISA in 60 sera of children not suffering from pneumonia. However, if only high titres (> 800) were regarded as indicative ofMycoplasma pneumoniae infection, a presumptive diagnosis could have been made in 42 of 73 single acute phase sera from patients. Comparable results were obtained with IHA. The diagnostic level of IgM antibodies was reached during the second week of the disease. It is concluded that examination of a single serum sample by either ELISA-IgM or IHA may assist in early diagnosis ofMycoplasma pneumoniae infection.     

Antibody-dependent cell-mediated cytotoxicity againstEscherichia coli O antigens

Antibodies againstEscherichia coli O antigen from rabbits immunized with formalin-killed bacteria were tested for cytotoxic capacity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay with human lymphocytes as effector cells and autologous papainized erythrocytes coated with O antigen as target cells. The cytotoxic titres were compared with the titres obtained with three methods of antibody quantitation. It was found that ADCC recorded antibodies with similar sensitivity as the enzyme-linked immunosorbent assay (ELISA) for IgG, but was much more sensitive than the ammonium sulphate precipitation (ASP) and indirect haemagglutination (IHA) usingβ-mercaptoethanol reduced sera. The ADCC titres were found to correlate very well with the titres obtained with ASP, ELISA and IHA for IgG but not for IgM, which is in accordance with a previous notion that ADCC is primarily mediated via IgG antibodies. ADCC should be considered as a possible immunopathologic mechanism in renal parenchymal damage in connection with urinary tract infections.     

An anti-human immunoglobulin M monoclonal antibody for detection of antibodies toToxoplasma gondii

An anti-human μ-chain monoclonal antibody, Tibi 82, was produced and tested for specificity by radioimmunoassay. Its reliability in detecting IgM antibodies toToxoplasma gondii was tested by two reverse immunosorbent methods (IgM-ISAGA and IgM-SPIHA) and the IgM fluorescent antibody test (IgM-IFA) on 400 sera. Whereas the results obtained with Tibi 82 and with two polyclonal reagents were highly correlated, the third commercial polyclonal reagent provided many false negative results. By standardizing IgM binding, Tibi 82 allowed the comparison of IgM-ISAGA with IgM-SPIHA on 100 sera: 17 % of the sera tested showed discrepancies due to the different toxoplasma antigens used. Although Tibi 82 facilitated the reading of results and enhanced sensitivity and specificity of the double-sandwich IgM-IFA method, the latter was still less sensitive than IgM-ISAGA with Tibi 82. Tests with the monoclonal antibody were consistently superior to tests with polyclonal antibodies.     

Intravenous immunoglobulin-mediated immunosuppression and the development of an IVIG substitute

Immunoglobulins are glycoproteins produced by the cells of the immune system. Their primary function is to protect the body from pathogenic infection. Moreover, a concentrated polyclonal mixture of immunoglobulin G (IgG), the so-called intravenous IgG (IVIG), has been used to treat various chronic and systemic disorders of the immune system. Studies on the effects of IVIG in autoimmune disease models have revealed that IgG Fc fragments confer protection against various autoimmune diseases. The identification of this IgG Fc immunomodulatory component is important for the development of IVIG substitutes. The focus of this review is to introduce one of the Fc regulatory entities and to provide a summary of the current knowledge of the putative general mechanisms underlying IVIG activity in vivo on the basis of these Fc fragments. We also address the recent insights into several approaches for the development of IVIG substitutes.     

Enzyme-linked immunosorbent assay for detection of solubleToxoplasma gondii antigen in acute-phase toxoplasmosis

Sixty-two sera from 51 patients with lymphadenopathy presumed to be due to acute-phase toxoplasmosis were tested for specific IgM class antibodies by both the immunofluorescence antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma antibody toToxoplasma gondii in sera were first dissociated in 3M NaSCN. Antigen attached to the solid phase was detected with enzyme-coupled IgG antibody toToxoplasma gondii. Neither hepatitis B surface antigen nor antigen ofMycoplasma pneumoniae, rubella, cytomegalovirus or herpes simplex virus interfered with this ELISA. Soluble antigen was detected in 13(30%) of 42 IgM-positive acute-phase toxoplasmosis patients and in only one of 20 sera cleared of IgM. None of an additional 44 IgM-negative patients with low IgG titres had a positive result in the antigen ELISA. Follow-up studies in four acute-phase toxoplasmosis patients showed that the soluble antigen cleared in all cases before the specific IgM antibodies. Simultaneous detection of IgM antibodies toToxoplasma gondii and soluble antigen would thus seem to indicate an early stage of the infection.     

Subcellular fractions in rubella immunoassays

Rubella virus-infected cells were fractionated by differential and sucrose gradient centrifugations. Rubella virus antigens distributed into all fractions but particulate material in the 100, 000×g pellet was shown to be enriched about two-fold for rubella virus antigen. Similarly, sucrose gradient fractions for rough endoplasmic reticulum and smooth cellular membranes were enriched for rubella virus antigens. The 100, 000 ×g pellet and the isolated cellular membranes proved to be useful when different fractions were used in solid-phase immunoassays for rubella virus-specific IgG or IgM. These fractions were equal in quality of the semipurified rubella virus preparations in the IgG assays but inferior to those in the IgM assays. However, simultaneous use of 35/25 % sucrose fractions from infected and non-infected cells reveals non-specific binding of IgM to the antigens and renders the IgM tests more specific for rubella virus.     

Mutational profiling of the RAS,PI3K,MET and b-catenin pathways in cancer of unknown primary: a retrospective study of the Hellenic Cooperative Oncology Group

Cancer of unknown primary origin (CUP) had a poor prognosis, determined by clinico-histological characteristics, partly due to the lack of insights on its biology. We screened tumour DNA from 87 patients with CUP for CTNNB1 (coding exons 2,3,4,5), MET (coding exon 18), PIK3CA (coding exons 9,20), KRAS (coding exons 1,2), BRAF (coding exon 15) gene mutations by using dd-sequencing and evaluated their impact on prognosis. Mutated gene incidences in the 87 CUP cases were: KRAS 11 (12.6 %), BRAF 5 (5.7 %), PIK3CA 8 (9 %), MET 6 (6.7 %) and CTNNB1 18 (20.7 %). Several mutations in the KRAS gene were not the commonly encountered mutations in other solid tumours. Activating mutations were observed in 10.2 % in KRAS, 4.5 % in BRAF, 6.6 % in PIK3CA, 4.5 % in MET, and 19.5 % in CTNNB1. Activating mutations in PIK3CA coding exon 9 were inversely correlated with MET coding exon 18 activating mutations (p = 0.036). MET activating mutations were prognostic for poor Progression-Free Survival (median PFS 5 vs 9 months, p = 0.009) and Overall Survival (median OS 7 vs 20 months, p = 0.005). The complex profile of either CTNNB1 or MET mutations also had an adverse prognostic significance (median OS 11 vs 21 months, p = 0.015). No other gene mutation exhibited prognostic significance. In multivariate analysis, poor performance status, male gender, visceral disease and adenocarcinoma histology, but not gene mutations, were independently associated with poor patient outcome. CTNNB1 gene mutations are frequent, and along with MET mutations have an adverse prognostic effect in patients with CUP.     

Magnolol Inhibits Tumor Necrosis Factor-α-Induced ICAM-1 Expression via Suppressing NF-κB And MAPK Signaling Pathways in Human Lung Epithelial Cells

Magnolol is a traditional Chinese medicine from the root and bark of Magnolia officinalis. It has long been used to treat anxiety, cough, headache and allergies, as well as a variety of inflammations. Lung inflammation is a key event in the pathogenesis of asthma and chronic obstructive pulmonary disease. The present study sought to examine the effects of magnolol on tumor necrosis factor (TNF)-α-induced upregulation of intercellular adhesion molecule-1 (ICAM-1), activation of the nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathway in cultured human pulmonary epithelial cells, and adhesion of human macrophage-like U937 cells to A549 cells. A549 cells were incubated with magnolol at 25 and 50 μmol/l. Then, 20 ng/ml TNF-α was used to activate the cells. Magnolol inhibited the growth of human pulmonary epithelial A549 cells in a dose- and time-dependent manner. Magnolol suppressed the adhesion of U937 cells to TNF-α-induced A549 cells. In cultured human pulmonary epithelial A549 cells, magnolol decreased TNF-α-induced upregulation of ICAM-1. Magnolol repressed TNF-α-induced activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in A549 cells by inhibiting phosphorylation of NF-κB, p38, extracellular signal-regulated kinase (ERK) 1/2, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). These findings support the hypothesis that magnolol inhibits the inflammatory process in lung epithelial A549 cells by suppressing the ICAM-1 and NF-κB and MAPK signaling pathways. Taken together, these results indicate that magnolol offers significant potential as a therapeutic treatment for inflammatory diseases of the lungs including asthma, sepsis, and chronic obstructive pulmonary disease.     

Use of hepatitis B core antigen produced inEscherichia coli to detect immunoglobulin M specific antibodies in an enzyme-linked immunosorbent assay

The antigenic activity of HBcAg produced inEschericha coli and HBcAg from human liver was compared in aμ-specific solid-phase antibody-capture assay for detection of anti-HBc-IgM. HBcAg from liver could be detected in dilutions up to 1∶3, HBcAg fromEscherichia coli in dilutions up to 1∶10, 000. Using HBcAg fromEschericha coli, sera from five patients with acute resolving hepatitis B and sera from four patients with actue hepatitis B who had developed chronic liver disease were tested for anti-HBc-IgM in ELISA. IgM fractions separated out of the same sera by immunoaffinity chromatography were tested for anti-HBc-IgM using a commercially available test. The results were in good agreement with those obtained by ELISA. Anti-HBc-IgM could be detected up to 900 days after onset of disease. Different groups of patients were tested for presence of anti-HBc-IgM in ELISA. Fifty-nine of 60 patients with acute hepatitis B were positive for anti-HBc-IgM at onset of illness. Ten of 16 patients with chronic aggressive hepatitis and seven of 23 HBsAg positive dialysis patients were also positive for anti-HBc-IgM, whereas only two of 12 patients with chronic persistent hepatitis and one of 15 HBsAg positive blood donors (“healthy” carriers of HBsAg) had detectable anti-HBc-IgM.     

Insulin is involved in transcriptional regulation of NKCC and the CFTR Cl− channel through PI3K activation and ERK inactivation in renal epithelial cells

It is is well known that insulin stimulates glucose transport and epithelial Na⁺ channel (ENaC)-mediated Na⁺ reabsorption; however, the action of insulin on Cl^? secretion is not fully understood. In this study, we investigated the action of insulin on Na⁺–K⁺–2Cl^? cotransporter (NKCC)-mediated Cl^? secretion in epithelial A6 cells. Interestingly, insulin treatment remarkably enhanced the forskolin-stimulated Cl^? secretion associated with an increase in apical Cl^? conductance by upregulating mRNA expression of both CFTR and NKCC, although insulin treatment alone had no effect on the basal Cl^? secretion or apical Cl^? conductance without forskolin application. We next elucidated a role of phosphoinositide 3-kinase (PI3K) in the insulin-induced enhancement of the Cl^? secretion, since insulin actually activated PI3K, resulting in activation of Akt, a downstream molecule of PI3K. LY294002 (a PI3K inhibitor) reduced the Cl^? secretion by suppressing mRNA expression of NKCC, whereas insulin still had a stimulatory action on mRNA expression of CFTR even in the presence of LY294002. On the other hand, we found that a MEK inhibitor (PD98059) further enhanced the insulin-stimulated CFTR mRNA expression and the Cl^? secretion in forskolin-stimulated A6 cells and that insulin induced slight, transient activation of ERK followed by significant inactivation of ERK. These observations suggest that: (1) insulin respectively upregulates mRNA expression of NKCC and CFTR through activation of PI3K and inactivation of ERK; (2) insulin signals on mRNA expression of NKCC and CFTR are not enough to stimulate transepithelial Cl^? secretion, but enhance the stimulatory action of cAMP on transepithelial Cl^? secretion.     

Piceatannol inhibits phorbol ester-induced expression of COX-2 and iNOS in HR-1 hairless mouse skin by blocking the activation of NF-κB and AP-1

Objectives

The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory activity of piceatannol (trans-3,4,3′,5′-tetrahydroxystilbene) in mouse skin in vivo.

Methods

Female HR-1 hairless mice were topically treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) with or without piceatannol pretreatment. Epidermal protein expression was assessed by Western blot analysis. The cyclooxygenase-2 (COX-2) expression was detected by immunohistochemistry. The DNA binding of nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) was examined by the electrophoretic mobility gel shift assay. The catalytic activity of IκBα kinase-β (IKKβ) was measured by in vitro kinase assay.

Results

Pretreatment with piceatannol attenuated TPA-induced expression of COX-2 and inducible nitric oxide synthase (iNOS) in mouse skin. Piceatannol diminished nuclear translocation and the DNA binding of NF-κB through the blockade of phosphorylation and subsequent degradation of IκBα. Piceatannol attenuated the catalytic activity of IKKβ and inhibited the phosphorylation of mitogen-activated protein (MAP) kinases in TPA-treated mouse skin. In addition, piceatannol decreased TPA-induced expression of c-Fos and the DNA binding of AP-1.

Conclusion

Piceatannol inhibits TPA-induced COX-2 and iNOS expression by blocking the activation of NF-κB and AP-1 via suppression of the IKKβ activity and phosphorylation of MAP kinases, which provides a mechanistic basis of its anti-inflammatory effects in mouse skin.     

Targeting CCR5 for anti-HIV research

Highly active antiretroviral therapy (HAART) is the only approach for human immunodeficiency virus (HIV) infection treatment at present. Although HAART is effective in controlling the progression of infection, it is impossible to eradicate the virus from patients. The patients have to live with the virus. Alternative ways for the cure of HIV infection have been investigated. As the major co-receptor for HIV-1 infection, C-C motif chemokine receptor 5 (CCR5) is naturally an ideal target for anti-HIV research. The first CCR5 antagonist, maraviroc, has been approved for the treatment of HIV infection. Several other CCR5 antagonists are in clinical trials. CCR5 delta32 is a natural genotype, conferring resistance to CCR5 using HIV-1 strains. Gene therapy research targeting this mutant has been conducted for HIV infection treatment. A Berlin patient has been cured of HIV infection by the transplantation of stem cells from a CCR5 delta32 genotype donor. The infusion of an engineered zinc finger nuclease (ZFN)-modified autologous cluster of differentiation 4 (CD4) T cells has been proved to be a promising direction recently. In this study, the anti-HIV research targeting CCR5 is summarized, including CCR5 antagonist development, stem cell transplantation, and gene therapy.     

Searching for the cause of the acquired immune deficiency syndrome

An outbreak of unexplained immune deficiency associated with opportunistic infection and Kaposi's sarcoma is occuring in the USA and other parts of the world. Affected individuals with what has come to be known as the acquired immune deficiency syndrome (AIDS) have a high mortality. Epidemiological features suggest the presence of a transmissable agent, but no responsible agent has yet been identified. Homosexual and bisexual men make up 75 % of these affected individuals. Cytomegalovirus, Epstein Barr and herpes simplex viruses, organisms that commonly affect male homosexuals, may produce some features of AIDS. Individually or collectively, however, they can not account for the emergence of a previously unrecognized clinical syndrome. Hepatitis B is prevalent in patients with AIDS and may play a role as a co-factor in the disease. The properties of a number of other known viruses may provide a model for the pathogenesis of some features of the AIDS immunodeficiency. Newly described simian acquired immune deficiency syndrome (SAIDS) is the best available animal model. In man, the retrovirus, human T-cell leukemia virus (HTLV) may play a role in AIDS. However, HTLV or any other known virus cannot yet be assumed to cause AIDS. It is likely that an as yet unrecognized agent is the key causative agent of AIDS.     

Dependence of cyclic-AMP induced relaxation on Ca2+ and calmodulin in skinned smooth muscle of guinea pigTaenia coli

cAMP (10^?6–10^?4 M) produced a dose-dependent relaxation of Ca²⁺-induced contraction in the guinea-pig taenia coli skinned with 1% Triton X-100. At 0.53 μM Ca²⁺ and 0.05 μM calmodulin (CaM), cAMP (10^?4 M) produced a maximal relaxation of 75% (pH 6.7; 25°C). Increasing Ca²⁺ (0.8 μM) or CaM (0.37 μM) reduced cAMP-induced relaxation to 25 and 5% respectively. At high CaM (5 μM), cAMP-induced relaxation could be completely inhibited by as low as 0.25 μM Ca²⁺. Furthermore, small increases in Ca²⁺ or CaM could effectively reverse the cAMP-induced relaxation in the continuous presence of cAMP. These results demonstrate that small modulations in the Ca²⁺-calmodulin activity have a strong effect on the ability of cAMP to produce a direct relaxing effect on the contractile proteins in skinned fiber. It is suggested that the effects of cAMP on the cellular mechanisms that lower cytoplasmic free Ca²⁺ concentration may act as the important determinants of the extent of the direct inhibitory effect of cAMP on the contractile elements. These two mechanisms may act in concert in this fashion to effect cAMP-induced relaxation in smooth muscle during β-adrenergic stimulation.     

Analysis of the interferon system in African patients with acquired immunodeficiency syndrome

Serum interferon and in vitro production of alpha and gamma interferon by peripheral blood leucocytes were examined in 21 African patients with acquired immunodeficiency syndrome (AIDS) and in 15 African patients with AIDS-related complex. Interferon was detected in the serum of 44 % of the patients with AIDS-related complex and in 70 % of the patients with full-blown AIDS, and was characterized as an acid-labile alpha interferon. When compared to healthy blood donors, the interferon response of peripheral blood leucocytes to Newcastle Disease virus was impaired in 7 of 12 patients with AIDS-related complex and in 16 of 20 AIDS patients (p<0.005). Also, production of gamma interferon following stimulation with phytohaemagglutinin was diminished in 5 of 11 patients with AIDS-related complex and in 13 of 17 patients with AIDS (p<0.005). A high correlation was observed between the presence of circulating interferon and decreased in vitro production of gamma interferon, but not of alpha interferon. These results suggest that the impairment of in vitro production of gamma interferon can be used as a preclinical marker of AIDS.     

Dietary Selenium Deficiency Exacerbates Lipopolysaccharide-Induced Inflammatory Response in Mouse Mastitis Models

Selenium (Se) is an essential micronutrient that plays a critical role in anti-inflammatory processes and antioxidant defense system. In this study, we investigated the effects of dietary selenium deficiency on lipopolysaccharide (LPS)-induced mastitis in mouse models. Se content in the liver was assessed by fluorescent atomic absorption spectrometry. Glutathione peroxidase (GPx) activity in the blood, myeloperoxidase (MPO) activity, tumor necrosis actor alpha (TNF-α), and interleukin (IL)-1β in the supernatant of the mammary tissue were determined according to the corresponding kits. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions were evaluated by Western blotting. The results showed that the Se-deficient mouse model was successfully replicated, and selenium deficiency exacerbated mammary gland histopathology, increased the expressions of TNF-α and IL-1β, and facilitated the activation of iNOS and COX-2 in LPS-induced mouse mastitis. In conclusion, our studies demonstrated that selenium deficiency resulted in more severe inflammatory response in LPS-induced mouse mastitis.     

The Big Five default brain: functional evidence

Recent neuroimaging studies have provided evidence that different dimensions of human personality may be associated with specific structural neuroanatomic correlates. Identifying brain correlates of a situation-independent personality structure would require evidence of a stable default mode of brain functioning. In this study, we investigated the correlates of the Big Five personality dimensions (Extraversion, Neuroticism, Openness/Intellect, Agreeableness, and Conscientiousness) and the default mode network (DMN). Forty-nine healthy adults completed the NEO-Five Factor. The results showed that the Extraversion (E) and Agreeableness (A) were positively correlated with activity in the midline core of the DMN, whereas Neuroticism (N), Openness (O), and Conscientiousness (C) were correlated with the parietal cortex system. Activity of the anterior cingulate cortex was positively associated with A and negatively with C. Regions of the parietal lobe were differentially associated with each personality dimension. The present study not only confirms previous functional correlates regarding the Big Five personality dimensions, but it also expands our knowledge showing the association between different personality dimensions and specific patterns of brain activation at rest.