An inhibition ELISA to determine alpha macroglobulin levels in mouse plasma

A sensitive method for quantifying mouse plasma alpha-macroglobulins (AM) using an inhibition ELISA is described. AM are important plasmaproteinase inhibitors that possibly act also as immunomodulatory molecules. The standard protocol develope in our experiments involves coating well with 10 µg/ml A2M in carbonate buffer, followed by incubation with a 1:1 (v/v) mixture of the plasma to be tested (diluted 1/1000) and goat anti-AM (diluted 1/1250). This is followed by further incubation, first with the enzyme-conjugated antibody and with the substrate prior to the reading of absorbance levels of the reaction products. Standard curve samples must be included in each plate, employing known amounts of the purified Murine Alpha-2-Macroglobulin (MuA2M) used for coating, with concentrations ranging from 0.001 to 10 µg/ml. Using test samples in triplicates and a 6-point standard curve in a single ELISA plate, 25 plasma samples can be tested accurately. The method offers an useful tool for establishing AM levelsin small samples of mouse plasma.

A α-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

α-glycerophosphate dehydrogenase (α-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

The balance between the production of tumor necrosis factor-alpha and interleukin-10 determines tissue injury and lethality during intestinal ischemia and reperfusion

A major goal in the treatment of acute ischemia of a vascular territory is to restore blood flow to normal values, i.e. to "reperfuse" the ischemic vascular bed. However, reperfusion of ischemic tissues is associated with local and systemic leukocyte activation and trafficking, endothelial barrier dysfunction in postcapillary venules, enhanced production of inflammatory mediators and great lethality. This phenomenon has been referred to as "reperfusion injury" and several studies demonstrated that injury is dependent on neutrophil recruitment. Furthermore, ischemia and reperfusion injury is associated with the coordinated activation of a series of cytokines and adhesion molecules. Among the mediators of the inflammatory cascade released, TNF-alpha appears to play an essential role for the reperfusion-associated injury. On the other hand, the release of IL-10 modulates pro-inflammatory cytokine production and reperfusion-associated tissue injury. IL-1beta, PAF and bradykinin are mediators involved in ischemia and reperfusion injury by regulating the balance between TNF-alpha and IL-10 production. Strategies that enhance IL-10 and/or prevent TNF-alpha concentration may be useful as therapeutic adjuvants in the treatment of the tissue injury that follows ischemia and reperfusion.

Importance of TNF-alpha in the course of acute infection with Trypanosoma cruzi: influence of its inhibition by pentoxifylline treatment

Infection of C3H/He mice with the Peruvian strain of Trypanosoma cruzi (Biodeme type I, Z2b), a macrophagotropic strain, determined severe parasitism of macrophages, necrosis of the spleen, and high host mortality. In the present study, pentoxifylline (PTX), an inhibitor of TNF-alpha was investigated on its action upon splenic necrosis, parasitemia and host survival. Immunohistochemical data suggested the importance of this cytokine in parasite destruction and decreasing of parasitemia, although paradoxically contributing to the high mortality of infected mice. Necrotic lesions involving several organs, specially the heart, in acute Chagas disease, are important aggravating factors, increasing cardiac morbidity. Advantage of inhibiting TNF-alpha action was herein investigated. Infected mice were divided into two groups: untreated (n = 24), and PTX treated mice (n = 25). PTX was administered in two daily doses of 30 mg/kg/bw, by intraperitoneal route. Normal controls either treated with PTX or saline were also included. Histopathology of the spleen and in situ immunolabeling of TNF-alpha, using anti-TNF-alpha monoclonal antibody, were performed. Necrotic areas were evaluated by morphometry. Mice treated with PTX showed a significant decrease of necrotic areas and diminution of TNF-alpha expression in spleen tissue, suggesting that PTX treatment could control TNF-alpha effects, and thus be used as an adjuvant in the treatment of acute Chagas' disease.

High-density lipoprotein prevents SAA-induced production of TNF-α in THP-1 monocytic cells and peripheral blood mononuclear cells

In this study, we evaluated whether human serum and lipoproteins, especially high-density lipoprotein (HDL), affected serum amyloid A (SAA)-induced cytokine release. We verified the effects of SAA on THP-1 cells in serum-free medium compared to medium containing human serum or lipoprotein-deficient serum. SAA-induced tumour necrosis factor-alpha (TNF-α) production was higher in the medium containing lipoprotein-deficient serum than in the medium containing normal human serum. The addition of HDL inhibited the SAA-induced TNF-α release in a dose-dependent manner. This inhibitory effect was specific for HDL and was not affected by low-density lipoprotein or very low-density lipoprotein. In human peripheral blood mononuclear cells, the inhibitory effect of HDL on TNF-α production induced by SAA was less pronounced. However, this effect was significant when HDL was added to lipoprotein-deficient medium. In addition, a similar inhibitory effect was observed for interleukin-1 beta release. These findings confirm the important role of HDL and support our previous hypothesis that HDL inhibits the effects of SAA during SAA transport in the bloodstream. Moreover, the HDL-induced reduction in the proinflammatory activity of SAA emphasizes the involvement of SAA in diseases, such as atherosclerosis, that are characterized by low levels of HDL.