Doppler ultrasound of hepatic blood flow for noninvasive evaluation of liver fibrosis compared with liver biopsy and transient elastography.

BACKGROUND: Accurate quantification of liver fibrosis is essential for therapeutic decision-making and follow-up of chronic liver diseases. AIMS: To optimize the quality of non-invasive assessment of liver fibrosis in patients with chronic hepatopathy we compared Doppler ultrasound with liver histology and transient elastography (TE). METHODS: In this prospective observational study, we performed Doppler ultrasound of hepatic blood vessels as well as TE in 125 patients who underwent liver biopsy for diagnostic work-up of hepatopathy. Hepatic venous flow was evaluated by determining resistance index (HVRI) of the right hepatic vein. Doppler and TE results were compared with histological staging, grading and degree of steatosis obtained by liver biopsy. RESULTS: HVRI showed a high reliability in predicting fibrosis stage FII or higher (AUROC 93.7 %, HVRI < 1.185; sensitivity 89.66 % and specificity 86.32 %) and was superior to TE. Neither steatosis nor inflammation had significant influence on HVRI-based estimation of fibrosis (1.45 ± 0.2; 1.26 ± 0.05; 1.06 ± 0.06; 0.87 ± 0.08; 0.46 ± 0.11 for F0-FIV, respectively). HVRI differed significantly in different stages of fibrosis. In contrast, portal vein and hepatic artery only showed significant changes in higher stages of fibrosis. Hepatic artery resistance index was elevated (0.67-0.74; p < 0.05); portal vein flow maximum and undulation were significantly reduced in higher fibrosis (p < 0.05 and p < 0.01, respectively). CONCLUSIONS: Hepatic blood flow analysis, especially HVRI, provides useful information during assessment of hepatopathy and is a reliable predictor of liver fibrosis stage FII or higher as part of the non-invasive diagnostic work-up and follow-up in chronic liver disease.

Lipopolysaccharide-like molecules derived from Wolbachia endobacteria of the filaria Onchocerca volvulus are candidate mediators in the sequence of inflammatory and antiinflammatory responses of human monocytes.

The majority of Onchocerca volvulus-infected persons show signs of cellular anergy, and long-time survival of adult and larval parasites in subcutaneous tissue is observed. The mechanisms leading to immunological hyporesponsiveness are poorly understood. Monocytes/macrophages represent a link between the innate and acquired immune system and are candidate cells to promote inflammatory and antiinflammatory processes. In the present study we have shown that products of microfilarial (O. volvulus) and adult (O. volvulus and O. ochengi) parasites affect monocytes in vitro. An early production of TNF-alpha by exposed monocytes was followed by the production of IL-10 and a reduced expression of HLA-DR and the costimulatory molecules B7-1 and B7-2, while other adhesion receptors remained unaffected. Downregulation of the functional membrane receptors failed to occur after treatment of the cells with anti-IL-10 antibodies. The engagement of CD14, a dominant membrane receptor on monocytes and major binding protein for lipopolysaccharides, was indicated by partial blocking of monocyte modulation by neutralizing antibodies to CD14 and by the antagonistic lipid A analog compound 406. Lipopolysaccharide-like molecules were detected in sterile products of O. volvulus stages which could originate from Wolbachia bacteria related to Gram-negative Rickettsiales, known to be abundant in the hypodermis and the female reproductive organs of O. volvulus. The present results indicate that the monocyte/macrophage may be a major target cell for immunomodulatory parasite-derived and intraparasitic, bacteria-derived molecules, thereby contributing to the host's cellular hyporesponsiveness.

Analysis of renal function in onchocerciasis patients before and after therapy.

The occurrence of renal abnormalities was investigated in patients with onchocerciasis in comparison to individuals without onchocerciasis in Guinea. Serum creatinine levels, excretion of urinary marker proteins, and kidney size by ultrasound were determined. A high prevalence of glomerular as well as tubular dysfunctions was observed; however, no association with onchocerciasis could be detected. We also hypothesized that patients with hyperreactive onchocerciasis might be prone to develop immune-mediated glomerular disorders; however, this could not be verified. Following treatment with ivermectin, a slight but significant increase in the excretion of urinary albumin and alpha1-microglobulin was seen five days after treatment in all treated patients, whereas levels of proteinuria were significantly higher five days after treatment only in patients with high microfilarial densities. Our results indicate that ivermectin can cause glomerular and tubular disturbances in patients with onchocerciasis; however, these are minor and do not seem to be clinically relevant.

Serum-dependent interaction of granulocytes with Onchocerca volvulus microfilariae in generalized and chronic hyper-reactive onchocerciasis and its modulation by diethylcarbamazine.

The adherence and cytotoxicity of granulocytes to microfilariae of Onchocerca volvulus were examined in vitro. Reactivity and modulation by diethylcarbamazine of isolated eosinophilic and neutrophilic granulocytes from patients with generalized and chronic hyper-reactive onchocerciasis (sowda or localized form) from endemic foci in Liberia were evaluated under varying serum conditions. In the presence of pooled sera from patients with generalized onchocerciasis granulocytes from both polar groups of patients exhibited similar adherence rates, whereas immobilization rates were higher for eosinophils than for neutrophils. In localized onchocerciasis, the use of autologous serum resulted in a significant decrease in adherence and immobilization rates for both eosinophils and neutrophils. After preincubation of eosinophils, but not of microfilariae, with diethylcarbamazine autologous serum-mediated adherence and cytotoxicity were enhanced to rates similar to those found with pooled serum from individuals with generalized onchocerciasis. These results suggest that granulocytes from both forms of onchocerciasis did not differ with respect to their anti-parasitic reactivity and that antibodies as well as additional serum factors appear to contribute to the functional activity of these effector cells. The findings indicate that predominantly eosinophils, compared to neutrophils, damage the larvae of O. volvulus and support earlier observations which suggest that diethylcarbamazine influences the effector cells rather than the parasite itself.

Urinary lysozyme phenotypes in monocytic leukemia.

A method of two-dimensional electrophoresis has been devised to allow the study of the electrophoretic mobility of urinary lysozyme. Three different phenotypes have been defined in a study of thirteen purified lysozymes obtained from different patients with monocytic leukemia.

Ultrastructural study of the interaction between eosinophilic granulocytes and third and fourth stage larvae of Onchocerca volvulus.

The interaction in vitro between eosinophil effector cells and third and fourth stage larvae of Onchocerca volvulus was studied by electron microscopy. The morphological observations demonstrated different mechanisms of attack of eosinophil cells that are dependent upon the time of incubation. Rapid adherence to the cuticle of the target, flattening, secretion of granule contents, vacuole formation and, finally, complete degranulation of the eosinophils were seen after incubation with third stage larvae and moulting stages. Alterations of the epicuticular and cuticular structures could be found near the attachment site of the cells. The eosinophils, however, showed no interactions with fourth stage larvae of this filarial parasite.

Serum levels of eosinophil cationic protein, eosinophil-derived neurotoxin and myeloperoxidase in infections with filariae and schistosomes.

The serum levels of three major granulocyte proteins were measured in patients with onchocerciasis, bancroftian filariasis and intestinal schistosomiasis and compared to controls from patients with malaria, Africans living in areas not endemic for these infections and healthy Germans. The investigation comprised the determination of the eosinophil granule proteins eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN/EPX), and the neutrophil/monocyte granule protein myeloperoxidase (MPO). ECP and EDN/EPX levels were found elevated only in the three helminth infections that are associated with eosinophilia, while MPO was found elevated in all tested disease groups. The levels of eosinophil granule proteins observed in the helminth diseases by far exceeded those described for bronchial asthma and atopic dermatitis. ECP, EDN/EPX and MPO serum levels reflect the ongoing disease and are related to functional activity of the respective leukopoetic system. ECP and EDN/EPX appear to be markers of the eosinophil effector system and MPO a marker of the neutrophil and/or monocyte/macrophage effector system. Significantly higher ECP levels in chronic hyperreactive onchodermatitis (sowda) versus generalized onchocerciasis seem to reflect an augmented degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates, indicating a more active mechanism of parasite clearance in sowda patients.

Modulatory effects of antifilarial drugs ivermectin, CGP 6140 and CGP 20376 on the oxidative burst of eosinophilic granulocytes.

Three recently introduced anthelmintic agents, the macrocyclic lactone, ivermectin, the amoscanate derivative CGP 6140 and the benzothiazole compound CGP 20376, were investigated for their in vitro modulatory effects on eosinophilic effector cells. The investigation comprised studies on the generation of the toxic oxygen intermediates superoxide anion and hydrogen peroxide which are major effector products of granulocytes. Eosinophils were obtained from 19 patients infected with the filarial parasite Onchocerca volvulus. Inhibitory effects on the generation of toxic oxygen intermediates were demonstrated for ivermectin and CGP 20376 at concentrations higher than 200 ng/ml (0.5 microM) and for CGP 6140 at concentrations higher than 1000 ng/ml (2.7 microM). An increased production of the reactive oxygen metabolites was demonstrated at low doses of ivermectin (20-40 ng/ml; corresponding to 0.02-0.04 microM) and CGP 6140 (40-100 ng/ml; 0.1-0.3 microM), respectively. The results reveal a dual, dose-dependent modulatory in vitro effect of the investigated anthelmintic drugs on the respiratory burst of eosinophilic effector cells indicating that these compounds may modulate host defense in vivo.

Eosinophils, eosinophil cationic protein and eosinophil-derived neurotoxin in serum and urine of patients with onchocerciasis coinfected with intestinal nematodes and in urinary schistosomiasis.

Eosinophils, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN/EPX), myeloperoxidase (MPO) and IgE were measured in blood, serum and/or urine in Schistosoma haematobium- and Onchocerca volvulus-infected Guineans and O. volvulus- and S. haematobium-negative Guineans coinfected or infected with intestinal nematodes. The number of eosinophils and levels of eosinophil granule proteins but not of MPO were found to be strongly elevated in all Africans as compared to European controls. The highest serum ECP and serum and urinary EDN/EPX levels were observed in the hyperreactive form of onchocerciasis (sowda). Onchocerciasis patients and O. volvulus-negative Africans coinfected or infected with intestinal nematodes (hookworm and/or Ascaris lumbricoides) revealed higher serum granule protein concentrations and/or absolute eosinophil counts and urinary ECP than those without nematode infections. Statistical differences between both sections were found for the absolute eosinophil counts and for serum EDN/EPX and IgE in generalized onchocerciasis, and for urinary ECP in sowda, indicating stimulation of the eosinophil potential of O. volvulus-positive patients by coexistent hookworm infection. This worm species, in contrast to A. lumbricoides, causes especially high eosinophil counts and EDN/EPX and IgE levels. From these results it is concluded that in nematode diseases, ECP and EDN/EPX levels reflect the degree of antigenic stimulation, eosinophil activation and eosinophil turnover rates. Serum ECP and serum and urinary EDN/EPX may, therefore, serve as parameters to monitor helminth infection. Urinary ECP may be a marker of eosinophiluria secondary to urogenital manifestation of S. haematobium. It is elevated in hyperreactive onchocerciasis activated by intestinal nematodes.

Cellular and subcellular localization of cationic leukocyte antigen (CLA) in human eosinophil granulocytes.

The cellular and subcellular localization of cationic leukocyte antigen (CLA) in human peripheral blood and tissue granulocytes was investigated by immunoenzymatic labeling and by immunoelectron microscopy. Human peripheral blood granulocytes from healthy individuals and from subjects with eosinophilia of varying etiology, as well as intravascular and/or perivascular granulocytes in skin biopsies taken from patients with generalized onchocerciasis, a skin disease caused by microfilariae of Onchocerca volvulus, were studied. Controlled indirect immunoenzymatic staining for CLA in cytospin preparations of buffy coat cells and in histological sections of skin biopsies revealed that this protein was exclusively found in the cytoplasm of eosinophil granulocytes. Furthermore, immunogold labeling coupled with electron microscopy showed that CLA was specifically localized within the matrix of both the small non-crystalloid-containing pale granules and the large crystalloid-containing secondary granules of peripheral blood and tissue eosinophils. No specific gold labeling was observed in other organelles of eosinophils, in neutrophils, basophils, lymphocytes or monocytes.

Improved Randolph stain for direct leukocyte differentiation and determination of total eosinophil count in a hemocytometer.

Identification and quantification of eosinophilic granulocytes are commonly performed indirectly by total leukocyte count and white cell differentiation in smears or cytocentrifuge preparations. Using a combination of four dyes, phloxine, Biebrich scarlet, methylene blue, and crystal violet, at 50-800 micrograms/ml, we have substantially improved an earlier method for differentiating leukocytes in a hemocytometer. This direct method allowed a rapid and reliable enumeration of eosinophils and their differentiation from neutrophils, lymphocytes and monocytes in peripheral blood and leukocyte fractions. The results obtained using this stain correlated with the leukocyte counts calculated from May-Grünwald-Giemsa stained smears in 100 patients with eosinophilia of various etiologies (r = 0.95; p < 0.01). This simple method is a useful improvement for eosinophil enumeration in field studies and biological experiments where the purity of cell suspensions must be evaluated without delay. The method cannot be substituted for the commonly used indirect technique which also allows the identification of other leukocyte lineages and their precursors.