Low levels of transforming growth factor-beta (TGF-beta) and reduced suppression of Th2-mediated inflammation in hyperreactive human onchocerciasis.

Th2-biased inflammation with eosinophilia and IgE production is a hallmark of helminth infections. It is pronounced in hyperreactive onchocerciasis patients ('sowda' or 'local form'), who efficiently kill microfilariae resulting in severe dermatitis and lymphadenitis. In contrast, hyporeactive patients ('generalised form') tolerate high microfilarial loads. This is thought to be mediated by regulatory CD4+ T cells and macrophages producing suppressive cytokines such as IL-10 and transforming growth factor-beta (TGF-ß). We investigated whether hyperreactivity was reflected by lower local TGF-ß production, analysing stable latent TGF-ß1 expression in onchocercomas, lymph nodes and skin from hyperreactive and hyporeactive patients by immunohistochemistry. TGF-ß expression was compared with that of IgE, IgG1, IgG4, and the antigen-presenting, CD4+ T cell-inducing MHC class II molecule HLA-DR. TGF-ß was weakly and less frequently expressed by various cell types in onchocercomas, skin and lymph nodes from hyperreactive compared to hyporeactive patients. This applied to reactions around living and dead adult worms as well as dead microfilariae. Antigen-presenting cells strongly expressed HLA-DR in both forms, but their numbers were reduced in hyperreactive nodules. Plasma cells produced more IgE and IgG1, but less of the anti-inflammatory antibody IgG4 in hyperreactive onchocercomas. In conclusion, hyperreactivity is linked with reduced local expression of TGF-ß, HLA-DR and IgG4, which might contribute to the insufficient down-regulation of inflammation via TGF-ß- and HLA-DR-induced regulatory lymphocytes.

COUP-TFII is regulated by high glucose in endothelial cells.

Diabetes mellitus is an important risk factor for cardiovascular diseases. Clinical evidence supports a link between hyperglycemia, endothelial dysfunction, and vascular disorders. However, the precise molecular mechanisms causing endothelial dysfunction in diabetic patients remain unclear. An interesting novel mediator could be chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays an essential role in glucose metabolism. COUP-TFII is known to be expressed in venous endothelial cells. In this study, we show COUP-TFII expression in human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells. HUVECs express glucose transporters 1, 3, 6, and 10, and the insulin receptor. Insulin in combination with glucose activates protein kinase B (PKB or Akt) phosphorylation via phosphoinositide 3-kinase (PI3-kinase). Short-term (60-240 min) stimulation of HUVECs with high glucose increased COUP-TFII expression independent of insulin. Long-term (48 h) stimulation of HUVECs with high glucose augmented expression of the insulin receptor and E-selectin, but downregulated COUP-TFII protein expression. Downregulation of COUP-TFII by shRNA leads to downregulation of E-selectin and upregulation of eNOS and glucose transporters. Our data suggest that COUP-TFII is regulated by glucose in a time- and dose-dependent manner in endothelial cells. COUP-TFII might affect endothelial function in a diabetic background.

Lack of interferon-gamma confers impaired neutrophil granulocyte function and imparts prolonged survival of adult filarial worms in murine filariasis.

We investigated the role of IFN-gamma in host defense during murine filariasis. Using the fully permissive infection of BALB/c mice with the rodent filaria Litomosoides sigmodontis, we show that interferon (IFN)-gamma is essential for encapsulation of adult filarial worms in inflammatory nodules and for normal worm clearance. IFN-gamma knockout (KO) mice had only one third of the nodules of wild-type mice but displayed a more than twofold increase in worm burden and increased microfilaremia. Neutrophil granulocytes, but not macrophages or eosinophils, appear to directly control worm load and nodule formation. Neutrophils, which we showed earlier to be essential for the encapsulation process in the thoracic cavity, where the worms reside, were diminished at this location in IFN-gamma KO compared to wild-type mice; they also displayed strongly reduced chemotactic and phagocytic activity compared to neutrophils of controls. This argues for a distinct defect in neutrophil activation accounting for the low formation of inflammatory nodules. Tumor necrosis factor-alpha, a major neutrophil-activating cytokine expressed by macrophages in the thoracic cavity around the worms, was highly induced in wild-type but absent in KO mice. Diminished activation of neutrophils seems to be a general hallmark of IFN-gamma KO mice, since neutrophils from uninfected KO mice also showed a reduction in chemotactic and phagocytic activity when induced by casein. In conclusion, these data are the first to define an IFN-gamma-dependent immune effector mechanism in murine filarial infection, i.e. neutrophil-mediated control of the adult worm load.

Altered donor and recipient Ly49+ NK cell subsets in allogeneic H-2d --> H-2b and H-2b --> H-2d bone marrow chimeras.

NK cells reject non-self hematopoietic bone marrow (BM) grafts via Ly49 receptor-mediated MHC class I-specific recognition and calibration of receptor expression levels. In this paper we investigated how Ly49+ subset frequencies were regulated dependent on MHC class I expression. The development of donor and host Ly49A+ (recognizes H-2Dd and H-2Dk ligands) and Ly49C/I+ (Ly49CBALB/c recognizes H-2Kb, H-2Kd, and H-2Dd, and Ly49CB6 recognizes only H-2Kb) NK cell frequencies were monitored for 120 days in murine-mixed allogeneic BM chimeras. C57BL/6 (H-2b) BM was transplanted into BALB/c (H-2d) mice and vice versa. Peripheral NK cell populations were examined every 5 days. Chimerism was found to be stable with 80-90% donor NK cells. In contrast to syngeneic controls reexpressing pretransplant patterns, donor and host NK cells revealed new and mainly reduced subset frequencies 55 days after allogeneic transplantation. Recipient NK cells acquired these later than donor NK cells. In H-2d --> H-2b chimeras Ly49A+, Ly49C/I+, and Ly49A+/Ly49C/I+ proportions were mainly diminished upon interaction with cognate ligands. Also in H-2b --> H-2d chimeras, Ly49A+ and Ly49A+/Ly49C/I+ subsets were reduced, but there was a transient normalization of Ly49C/I+ proportions in the noncognate host. After 120 days all subsets were reduced. Therefore, down-regulation of developing Ly49A+ and Ly49C/I+ chimeric NK cell frequencies by cognate ligands within 7-8 wk after BM transplantation may be important for successful engraftment.

Ivermectin influence on the mast cell activity in nodules of onchocerciasis patients.

Onchocercal nodules were stained immunohistochemically using antibodies specific for human mast cells and IgE to elucidate the localization and frequency of mast cells after a single oral dose of 150 microg/kg ivermectin. Tryptase-and chymase-positive mast cells occurred predominantly in mixed inflammatory infiltrates and perivascularly, and never adhered to adult worms or microfilariae. Up to three days after ivermectin, mast cells and IgE-positive cells were markedly increased in the capsular area of nodules containing female worms with embryos and microfilariae compared to untreated nodules. In the centre of these nodules, around the adult Onchocerca volvulus, we found many tryptase-positive cells. More mast cells were IgE-positive than in untreated nodules, equalling the number of tryptase-positive mast cells. There was a clear correlation between the appearance of mast cells and the attacks on damaged microfilariae by eosinophils and macrophages and in the vicinity of adult worms by neutrophils that occur soon after ivermectin treatment. Onchocercomata harbouring female worms with oocytes only revealed, after all treatment intervals, the same mast cell numbers as untreated nodules. In conclusion, during the first three days after administration, ivermectin produces increased numbers of mast cells in nodules harbouring females with embryos and microfilariae, probably as part of an allergic reaction to the attacked microfilariae. Four to 19 days after ivermectin the number of mast cells in the entire nodule is no longer elevated.

Mast cells in onchocercomas from patients with hyperreactive onchocerciasis (sowda).

In onchocerciasis, variations of the host's immune responsiveness produce a spectrum of clinical manifestations ranging from the common generalized to the rare hyperreactive form (sowda). For further characterization of the immune response, the localization and frequency of mast cells in onchocercomas from untreated and ivermectin-treated patients with hyperreactive onchocerciasis from Liberia and the Yemen were analysed and compared to the generalized form by immunohistochemistry with antibodies specific for human mast cell tryptase and chymase, histamine and IgE. The nodules were selected with special regard to only one pair of live, microfilariae-producing Onchocerca volvulus. Throughout the nodular tissue of the hyperreactive form, mast cells accumulated in the strong inflammatory infiltrates, especially near eosinophils and around cellular attacks on microfilariae as well as perivascularly. Their number was significantly higher in the whole nodular tissue compared to the generalized form. The highest numbers occurred in the nodule centre. Mast cells carried IgE and appeared activated. No mast cells were observed in the cystic parts or attached to adult worms or microfilariae. In onchocercomas, 1 and 3 days after treatment with ivermectin, microgranuloma formation by eosinophils and macrophages around damaged microfilariae was enhanced and accompanied by numerous mast cells. Attacks of neutrophils were also pronounced, but attacks by mast cells were not observed. In conclusion, hyperreactivity against microfilariae in onchocercomas clearly correlates with a strong mastocytosis and IgE production parallel to tissue eosinophilia.

Mast cell distribution in nodules of Onchocerca volvulus from untreated patients with generalized onchocerciasis.

Onchocercomata with a defined worm population were analysed to elucidate the distribution of mast cells. Nodules with live females were classified according to the presence or absence of microfilariae. Immunohistochemical staining was performed using antibodies specific for mast cells or IgE. Mast cells appeared singly or in diffuse accumulations perivascularly and in inflammatory infiltrates between adult Onchocerca volvulus and in the capsular area. No mast cells were detected in cystic parts. Only few, scattered mast cells were found in the fibrous zone around the adult worm. They were increased with stronger infiltration and hence, related to the inflammatory cells. Mast cells were never localized directly at adult worms or microfilariae. A correlation of the mast cell distribution to the occurrence of eosinophils was observed regarding higher numbers of mast cells and eosinophils in nodules with microfilariae-producing females. Nodules with single males revealed higher numbers of mast cells than nodules with non-producing females, although both contained very few eosinophils. Onchocercomata with dead worms contained significantly more mast cells than those with live filariae. In conclusion, the localization and frequency of mast cells is contingent on the vitality and productivity of the worms and therefore, indirectly and directly on the release of O. volvulus antigens.

Anesthetic cutoff in cycloalkanemethanols. A test of current theories.

BACKGROUND: N-alkanols containing up to 12 carbons are anesthetic; however, those with more than 12 carbons are not. This phenomenon has been termed cutoff. Lipid disordering theories of anesthesia suggest that cutoff occurs because the alkyl chains of long-chain alcohols approach the length and shape of the lipids of neuronal membranes and, therefore, intercalate into membranes without perturbing them. Protein theories suggest that cutoff occurs because the size of long-chain alcohols exceeds that of a protein binding site having finite dimensions. These theories were tested with a new series of alcohols, the cycloalkanemethanols, c(CnH2n-1).CH2.OH. METHODS: Anesthetic potency was measured in Rana pipiens tadpoles using the reversible loss of righting reflexes as the endpoint. The change in order parameter induced by cycloalkanemethanols and n-alkanols in lipid bilayers made of egg phosphatidylcholine and cholesterol was measured with electron paramagnetic resonance spectroscopy. RESULTS: On ascending the series from cyclopropanemethanol (EC50 = 54 +/- 3.2 mM) to cycloundecanemethanol (EC50 = 7.0 +/- 0.12 microM) anesthetic potencies first increased exponentially but then decreased sharply at cyclododecanemethanol (EC50 = 13 +/- 0.2 microM). Cyclotetradecanemethanol was found not to cause anesthesia in tadpoles, even after 48 h of exposure, although saturated solutions shifted the dose-response curve of octanol from 66 +/- 2.6 to 47 +/- 2.8 microM. A linear loss in the ability to disorder lipid bilayers was observed on ascending both alcohol series such that cyclotetradecanemethanol and n-tridecanol actually increased bilayer order. CONCLUSIONS: Molecular length does not correlate with anesthetic cutoff in these two alcohol series. Cutoff is predicted by the ability of both series of alcohols to disorder lipid bilayers and correlates with their molecular volume.