Interleukin-12 Enhances Antifungal Activity of Human Mononuclear Phagocytes against Aspergillus fumigatus: Implications for a Gamma Interferon-Independent Pathway

The potential of recombinant human interleukin-12 (IL-12) to enhance the capacity of human monocytes (MNC) to elicit an oxidative burst and damage hyphae of Aspergillus fumigatus was investigated. Incubation of peripheral blood mononuclear cells (PBMC) from healthy adults with 10 to 100 ng of IL-12/ml at 37 degrees C for 2 to 3 days enhanced the production of superoxide anion (O2-) in response to phorbol myristate acetate (PMA) (P = 0.04) and unopsonized A. fumigatus hyphae (P = 0.03) and further enhanced hyphal damage (P = 0.009). Anti-gamma interferon (anti-IFN-gamma) blocked secretion of IFN-gamma by IL-12-treated PBMC but did not inhibit IL-12-induced O2- production by these cells in response to PMA. In addition, IL-12-treated elutriated MNC secreted no IFN-gamma or tumor necrosis factor alpha but exhibited enhanced O2- production compared to controls (P = 0.013). These findings demonstrate that IL-12 augments oxidative antifungal activities of MNC via an IFN-gamma-independent route, suggesting a novel pathway of IL-12 action in antifungal defense.     

抗重组人肿瘤坏死因子-α(rHTNF-α)单克隆抗体特性及功能的研究

应用杂交瘤技木制备抗重组人肿瘤坏死因子-α单克隆抗体对于r-HTNF-α的纯化和TNF-α分子的抗原性及功能的研究将是一种主要工具。本实验对一组抗rHTNF-α单克隆抗体的特性和功能进行了研究。Western blotting结果表明Z_4、Z_8、Z_(12)、Z_ (20)、Z_(21)、B_3和E_6 抗体特异性地识别分子量为17000道尔顿的TNF-α它们与rIL-1、rIL-2、rIFNγ、rIFNα、和E coli菌裂解液无交叉反应。竞争性ELISA结果证实7种抗体分别识别TNF-α分子上5个不同的抗原决定簇。其中4个抗体可以识别TNF-α活性中心部位。两个抗体还可以识别天然TNF-α分子。     

Lipoprotein(a) Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production by Human Mononuclear Cells

Lipoproteins can bind lipopolysaccharide (LPS) and decrease LPS-stimulated cytokine production. Lipoprotein(a) [Lp(a)] was as potent as low-density lipoproteins (LDL) in inhibiting LPS-stimulated tumor necrosis factor synthesis by human mononuclear cells. The kinetics of LPS inhibition by Lp(a) was similar to that of LDL. This suggests that circulating Lp(a) may be an important factor determining the amplitude of the response to LPS in humans.     

High Producing Tumor Necrosis Factor Alpha Gene Alleles in Protection against Severe Manifestations of Dengue

Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. Methods: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. Results: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3''UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. Conclusion: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.     

Bikunin Inhibits Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Induction in Macrophages

Bikunin, a Kunitz-type protease inhibitor, exhibits anti-inflammatory activity in protection against cancer and inflammation. To investigate the molecular mechanism of this inhibition, we analyzed the effect of bikunin on tumor necrosis factor alpha (TNF-α) production in human peripheral mononuclear cells stimulated by lipopolysaccharide (LPS), an inflammatory inducer. Here, we show the following results. (i) LPS induced TNF-α expression in time- and dose-dependent manners through phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. (ii) Bikunin inhibits LPS-induced up-regulation of TNF-α protein expression in a dose-dependent manner, reaching 60% inhibition at the highest doses of bikunin tested (5.0 μM). (iii) Inhibition by bikunin of TNF-α induction correlates with the suppressive capacity of ERK1/2, JNK, and p38 signaling pathways, implicating repressions of at least three different signals in the inhibition. (iv) Bikunin blocks the induction of TNF-α target molecules interleukin-1β (IL-1β) and IL-6 proteins. (v) Bikunin is functional in vivo, and this glycoprotein blocks systemic TNF-α release in mice challenged with LPS. (vi) Finally, bikunin can prevent LPS-induced lethality. In conclusion, bikunin significantly inhibits LPS-induced TNF-α production, suggesting a mechanism of anti-inflammation by bikunin through control of cytokine induction during inflammation. Bikunin might be a candidate for the treatment of inflammation, including septic shock.     

Antifungal activity of endophytic fungi from Cupressaceae against human pathogenic Aspergillus fumigatus and Aspergillus niger

Aspergillus is a fungal genus that strongly affects health of humans, animals, and plants worldwide. Endophytes are now widely considered as a rich source of bioproducts with potential uses in medicine, agriculture, and bioindustry. Cupressaceae plant family hosts a variety of bioactive ascomycetous endophytes. In this study, antifungal activity of a number of such endophytes were investigated against human pathogenic fungi Aspergillus fumigatus and Aspergillus niger. To this end, 16 superior bioactive endophytic fungi from Cupressaceae were used, including Alternaria alternata, Alternaria pellucida, Ascorhizoctonia sp., Aspergillus fumigatus, Aspergillus niger, Aurobasidium sp., Cladosporium porophorum, Fusarium oxysporum, Penicillium viridicatum, Phoma herbarum, Phoma sp., Pyrenochaeta sp., Trichoderma atroviride, Trichoderma atroviride and Trichoderma koningii. In vitro bioassays indicated anti-Asperilli activity of the endophytic fungi in dual cultures. Most notably, Trichoderma koningii CSE₃₂ and Trichoderma atroviride JCE₃₃ showed complete growth inhibition of both A. niger and A. fumigatus, within 3 to 7 days. Also, volatile compouds (VOCs) of T. koningii CSE₃₂ and T. atroviride JCE₃₃ exhibited 33–100% growth inhibition of A. niger, whithin 3 days. Moreover, on the day 7, growth of A. niger was less affected than that of A. fumigatus. In general, it appears that there is a direct relationship between the exposure time and the inhibitory activity of endophytes VOCs on the growth of target Aspergillus species. Furthremore, the extracellular secondary metabolites (SMs) of four selected fungal endophytes exhibited anti-Aspergillus activity at all treatment levels as shown by Agar-diffusion assay. SMs from T. koningii CSE₃₂ and Pyrenochaeta CSE₁₃₄ showed strongest activities against A. niger, and SMs from T. koningii CSE₃₂ and F. oxysporum CAE₁₄ showed strongest activities against A. fumigatus. In conclusion, given the globally recognized issue of antibiotic resistance and the urge to discover new antimicrobial substances, our findings provide new insights into the potential use of Cupressaceae's endophytic fungi in antifungal-based drug discovery programs.     

Effects of Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha on Trypanosoma cruzi Trypomastigotes

We have previously shown that the addition of exogenous granulocyte-macrophage colony-stimulating factor (GM-CSF) to nonactivated mouse peritoneal macrophages (MPM) limits Trypanosoma cruzi infections in vitro (E. Olivares Fontt and B. Vray, Parasite Immunol. 17:135–141, 1995). Lower levels of infection were correlated with a higher level of production of tumor necrosis factor alpha (TNF-α) in the absence of nitric oxide (NO) release. These data suggested that GM-CSF and/or TNF-α might have a direct parasitocidal effect on T. cruzi trypomastigotes, independently of NO release. To address this question, T. cruzi trypomastigotes were treated with recombinant murine GM-CSF (rmGM-CSF), recombinant murine TNF-α (rmTNF-α), or both cytokines in a cell-free system. Treatment with rmGM-CSF but not rmTNF-α caused morphological changes in the parasites, and most became spherical after 7 h of incubation. Both cytokines exerted a cytolytic activity on the trypomastigotes, yet the trypanolytic activity of rmTNF-α was more effective than that of rmGM-CSF. Viable rmGM-CSF- and rmTNF-α-treated parasites were less able to infect MPM than untreated parasites, and this reduction in infectivity was greatest for rmGM-CSF. Treatments with both cytokines resulted in more lysis and almost complete inhibition of infection. The direct parasitocidal activity of rmTNF-α was inhibited by carbohydrates and monoclonal antibodies specific for the lectin-like domain of TNF-α. Collectively, these results suggest that cytokines such as GM-CSF and TNF-α may directly control the level of T. cruzi trypomastigotes at least in vitro and so could determine the outcome of infection in vivo.     

Expression of Interleukin-5 and Tumor Necrosis Factor Alpha in Cervical Carcinoma

Interleukin-5 (IL-5) levels were significantly higher in vaginal washing fluids from patients with cervical carcinoma than in those from patients with carcinoma in situ and controls. Tumor necrosis factor alpha levels did not differ among the three groups. Detection of IL-5 in cervical secretions may be a useful marker for evaluating aggressive local immune response in cervical carcinoma.Cellular immune response mediated by cytokines is the main defense against tumors related to cervical carcinogenesis. Previous studies have suggested that decreased T helper 1 (Th1) and increased Th2 responses are associated with cervical carcinogenesis (2, 3, 5, 6, 8). However, Th1 and Th2 cytokines, such as interleukin-1β (IL-1β), IL-10, IL-12, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta, were elevated in cervicovaginal washings from patients with cervical carcinoma (12). Another study reported that the concentrations of TNF-α and IL-10 were increased in cases of cervical intraepithelial neoplasia (1). Although each study was different in terms of increased cytokine profiles, the results reveal that both Th1 and Th2 cytokines are increased in cervical carcinoma, which differs from previous reports of a shift toward a Th2 cytokine pattern during cervical carcinogenesis.To date, most studies have focused on the cytokine profiles of the systemic immune response by analysis of peripheral blood. This study was designed to evaluate the cytokine secretion profiles for the Th2 cytokine IL-5 and the Th1 cytokine TNF-α in cervicovaginal secretions. In addition, we aimed to verify whether the levels of cytokines were related to eosinophil counts and human papillomavirus (HPV) DNA titers.Women with abnormal cervical cytology who had been referred to the Women''s Cancer Clinics of Severance Hospital between May 2006 and November 2006 were included. For all women, a cervical biopsy and HPV sampling were performed. Informed consent was obtained from each patient prior to enrollment. We recruited women who were diagnosed histologically with cervical carcinoma (n = 20) or carcinoma in situ (CIS) of the uterine cervix (n = 6). Women histologically diagnosed with chronic nonspecific inflammation were recruited for the control group (n = 10) ( Table ​Table11).

TABLE 1.

Demographic and clinical characteristics of patients in the control, CIS, and cervical carcinoma groups

Carrageenan Primes Leukocytes To Enhance Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Production

We have previously reported that pretreatment with carrageenan (CAR) enhances lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha) production in and lethality for mice. Whole blood cultured in vitro was used to show that CAR pretreatment results in about a 200-fold increase in LPS-induced TNF-alpha production. CAR by itself did not induce TNF-alpha production. However, CAR-treated cultured medium sensitized whole blood to make more LPS-induced TNF than did saline-treated cultured medium in vitro. It was also demonstrated that CAR pretreatment increases TNF-alpha mRNA levels of both blood cells and peritoneal exudate cells, but not of bone marrow cells. Immunoelectron microscopic analysis revealed that polymorphonuclear leukocytes and macrophages are TNF-alpha-producing cells in CAR-treated mice. In CAR-treated mice, TNF-alpha was seen early after LPS injection in leukocytes in hepatic sinusoids and on the surfaces of endothelial cells. TNF-alpha was also detected late after LPS injection in hepatocytes which become edematous. These results suggest that CAR primes leukocytes to produce TNF-alpha in response to LPS and that they play an important role in the pathogenesis of liver injury.     

Soluble Tumor Necrosis Factor Alpha Receptors in Sera from Leprosy Patients

Serum levels of soluble tumor necrosis factor alpha receptor I (sTNF-RI) were elevated in patients with lepromatous (LL) reactional-state type II leprosy, and sTNF-RII levels were increased in patients with full tuberculoid (TT) or LL type II leprosy. The sTNF-R in sera from patients with type II leprosy, but not other forms of leprosy, inhibited recombinant TNF cytolytic activities in vitro. This suggests that sTNF-R regulatory activities are partially impaired in patients with leprosy.     

Enhancement of Lipopolysaccharide-Induced Neutrophil Oxygen Radical Production by Tumor Necrosis Factor Alpha

Although tissues become exposed to both exogenous and endogenous cell-activating mediators during infection, there is little appreciation of the effects of subjecting cells to multiple mediators. We examined the hypothesis that the response of neutrophils to bacterial lipopolysaccharide (LPS) is significantly altered in the presence of the endogenous mediator tumor necrosis factor alpha (TNF). The data showed that human neutrophils pretreated with TNF for 10 to 30 min, displayed significantly enhanced superoxide production in response to LPS (from either Escherichia coli K-235 or E. coli 0127:B8), measured as lucigenin-dependent chemiluminescence (CL), seen as an increase in the initial peak rate as well as the total CL accumulated over the incubation period. TNF amplified the response to LPS at 1 to 100 U of TNF/10⁶ neutrophils and was able to enhance the response to a wide range of concentrations of LPS (0.01 to 1,000 ng/ml). The TNF-induced increase in the LPS response was paralleled by an increase in LPS binding to the neutrophils, which could be abrogated by an anti-CD14 monoclonal antibody. The results demonstrate that TNF significantly increases the LPS-induced release of oxygen radicals in neutrophils through the upregulation of cell surface CD14.     

Shedding of Tumor Necrosis Factor Receptor 1 Induced by Protein A Decreases Tumor Necrosis Factor Alpha Availability and Inflammation during Systemic Staphylococcus aureus Infection

Staphylococcus aureus infections are an important public health concern due to their increasing incidence and high rates of mortality. The success of S. aureus as a pathogen is highly related to its enormous capacity to evade the host immune response. The critical role of tumor necrosis factor alpha (TNF-α) in the initial host defense against systemic staphylococcal infection has been demonstrated in experimental models and may partially explain the lack of significant benefits observed in clinical trials attempting to neutralize this cytokine in septic patients. S. aureus protein A plays a key role in regulating inflammation through its ability to bind and signal through the TNF-α receptor 1 (TNFR1). In this study, we demonstrate that S. aureus, via protein A-mediated signaling, induces early shedding of TNFR1, which precedes the secretion of TNF-α in vitro and in vivo. The results obtained using a protein A-deficient mutant and tnfr1^−/− mice strongly suggest that the increased levels of soluble TNFR1 present during experimental S. aureus infection may neutralize circulating TNF-α and impair the host inflammatory response. Early shedding of TNFR1 induced by protein A may constitute a novel mechanism by which S. aureus subverts the host immune response.     

High Serum Interleukin-10 and Tumor Necrosis Factor Alpha Levels in Chronic Paracoccidioidomycosis

In patients with chronic paracoccidioidomycosis (n = 10), levels of tumor necrosis factor alpha, interleukin-10, and interleukin-2 in serum, measured by enzyme-linked immunosorbent assay (in picograms per milliliter, as mean ± standard error of the mean), were higher than in normal controls (n = 8): 186 ± 40 versus 40 ± 7 (P < 0.05), 203 ± 95 versus 20 ± 8 (P = 0.001), and 96.3 ± 78.57 versus 1.19 ± 1.19 (P = 0.045), respectively. Gamma interferon and interleukin-4 levels were similar in patients and controls.     

Salmonella Flagellin Induces Tumor Necrosis Factor Alpha in a Human Promonocytic Cell Line

During infection of the gastrointestinal tract, salmonellae induce cytokine production and inflammatory responses which are believed to mediate tissue damage in the host. In a previous study, we reported that salmonellae possess the ability to stimulate tumor necrosis factor alpha (TNF-α) accumulation in primary human monocytes, as well as in the human promonocytic cell line U38. In this model system, cytokine upregulation is not due to lipopolysaccharide but is mediated by a released protein. In the present study, TnphoA transposon mutagenesis was used to identify the TNF-α-inducing factor. A mutant Salmonella strain which lacks the ability to induce TNF-α was isolated from a TnphoA library. Genetic analysis of this mutant demonstrated that the hns gene has been interrupted by transposon insertion. The hns gene product is a DNA-binding protein that regulates the expression of a variety of unrelated genes in salmonellae. One of the known targets of histone-like protein H1 is flhDC, the master operon which is absolutely required for flagellar expression. Analysis of other nonflagellated mutant Salmonella strains revealed a correlation between the ability to induce TNF-α and the expression of the phase 1 filament subunit protein FliC. Complementation experiments demonstrated that FliC is sufficient to restore the ability of nonflagellated mutant Salmonella strains to upregulate TNF-α, whereas the phase 2 protein FljB appears to complement to a lesser extent. In addition, Salmonella FliC can confer the TNF-α-inducing phenotype on Escherichia coli, which otherwise lacks the activity. Furthermore, assembly of FliC into complete flagellar structures may not be required for induction of TNF-α.     

静脉溶栓治疗急性心肌梗死血浆TNF-а动态变化及意义

目的观察急性心肌梗死(AMI)再灌注治疗后血浆肿瘤坏死因子-а(TNF-а)的动态变化并评价其临床意义.方法对48例发病6小时内的AMI患者进行溶栓治疗,用放免法测定患者溶栓前、后0.5、1、2、4、12、48小时及1周血浆TNF-а及肌酸磷酸激酶(CPK)的浓度.结果 48例患者中36例再通,12例未通.溶栓前两组TNF-а浓度都大于正常值3倍.溶栓后 TNF-а于未通组48小时出现峰值(25.8±13.4 ng/ml),再通组无高峰,除峰值外两组无差异.结论 AMI患者血浆TNF-а动态曲线能反映AMI早期炎症情况.AMI炎症高峰在51小时,再灌注后TNF-а高峰不显.溶栓再灌注挽救心肌而减轻的炎症反应程度足以抵消再灌注炎症损伤.     

静脉溶栓治疗急性心肌梗死血浆TNF—a动态变化及意义

目的　观察急性心肌梗死 (AMI)再灌注治疗后血浆肿瘤坏死因子 -а(TNF -а)的动态变化并评价其临床意义 .方法　对 48例发病 6小时内的AMI患者进行溶栓治疗 ,用放免法测定患者溶栓前、后 0 .5、1、2、4、12、48小时及1周血浆TNF -а及肌酸磷酸激酶 (CPK)的浓度 .结果　 48例患者中 3 6例再通 ,12例未通 .溶栓前两组TNF -а浓度都大于正常值 3倍 .溶栓后TNF -а于未通组 48小时出现峰值 (2 5.8± 13 .4ng/ml) ,再通组无高峰 ,除峰值外两组无差异 .结论　AMI患者血浆TNF -а动态曲线能反映AMI早期炎症情况 .AMI炎症高峰在 51小时 ,再灌注后TNF -а高峰不显 .溶栓再灌注挽救心肌而减轻的炎症反应程度足以抵消再灌注炎症损伤     

Diagnostic Implications of Antigen-Induced Gamma Interferon, Nitric Oxide, and Tumor Necrosis Factor Alpha Production by Peripheral Blood Mononuclear Cells from Mycobacterium bovis-Infected Cattle

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-γ), nitric oxide (NO), and tumor necrosis factor alpha (TNF-α) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-γ, NO, and TNF-α responses. Infection-specific increases in NO, but not in IFN-γ or TNF-α, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-γ, NO, and TNF-α responses in antigen-stimulated cells from cattle receiving 10⁵ CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10³ CFU of M. bovis organisms. The NO response, but not the IFN-γ and TNF-α responses, was influenced by infective strains of M. bovis. The TNF-α, NO, and IFN-γ responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-α, like IFN-γ, may prove useful as indices for the diagnosis of bovine tuberculosis.     

Mycobacterium marinum SecA2 Promotes Stable Granulomas and Induces Tumor Necrosis Factor Alpha In Vivo

SecA2 is an ATPase present in some pathogenic Gram-positive bacteria, is required for translocation of a limited set of proteins across the cytosolic membrane, and plays an important role in virulence in several bacteria, including mycobacteria that cause diseases such as tuberculosis and leprosy. However, the mechanisms by which SecA2 affects virulence are incompletely understood. To investigate whether SecA2 modulates host immune responses in vivo, we studied Mycobacterium marinum infection in two different hosts: an established zebrafish model and a recently described mouse model. Here we show that M. marinum ΔsecA2 was attenuated for virulence in both host species and SecA2 was needed for normal granuloma numbers and for optimal tumor necrosis factor alpha response in both zebrafish and mice. M. marinum ΔsecA2 was more sensitive to SDS and had unique protrusions from its cell envelope when examined by cryo-electron tomography, suggesting that SecA2 is important for bacterial cell wall integrity. These results provide evidence that SecA2 induces granulomas and is required for bacterial modulation of the host response because it affects the mycobacterial cell envelope.     

Tumor Necrosis Factor Alpha Receptor I Is Important for Survival from Streptococcus pneumoniae Infections

Tumor necrosis factor alpha (TNF-α) is important in resistance to various microorganisms and provides signals to the target cells through two different receptors, TNF-α receptor I (TNFRI) (p55 receptor) and TNFRII (p75 receptor). To delineate the significance of the two different signaling pathways in resisting infections with extracellular bacteria, we examined the resistance of mice to Streptococcus pneumoniae (serotype 6B). TNF-α needs to be present early in infections, since one injection of wild-type mice with anti-TNF-α leads to an increased susceptibility of these mice to S. pneumoniae. TNF-α signaling through the p55 receptor (but not the p75 receptor) is crucial in resisting S. pneumoniae infections, because intraperitoneal injection of 100 CFU/mouse killed p55-deficient mice by day 2 of infection, whereas 1,000,000 CFU/mouse was needed to kill half of the control mice. p55-deficient mice do not show evidence of a deficient acute-phase response. All three types of mice (p55 deficient, p75 deficient, and normal) showed comparable rises in the levels of two acute-phase proteins (serum amyloid P and C3) at 24, 48, and 72 h after the experimental infections, and all of the mice showed comparable influxes of neutrophils to the site of infection. Finally, it was demonstrated that p55-deficient mice can be protected from the lethal effects of S. pneumoniae infection by injection of antibodies specific for S. pneumoniae polysaccharide capsule.