Human peripheral blood hypodense eosinophil proteins are more labeled with 32P than the normodense eosinophil proteins.

Peripheral blood eosinophils from patients with atopic dermatitis and normal healthy controls were isolated on a Percoll gradient and were incubated with [32P]orthophosphoric acid. After stopping the reaction, SDS/PAGE was performed and autoradiographs were prepared to determine the incorporation of 32P into proteins. Eosinophils developed at least 14 protein bands below 66.2 kDa by SDS/PAGE and the differences of the protein staining patterns between hypodense and normodense eosinophils were present. In the autoradiographs five distinct radioactive bands were observed below 31 kDa. 32P incorporation into the bands of hypodense eosinophils was stronger than that of normodense eosinophils, suggesting possible involvement of protein phosphorylation in the activation process of human eosinophils.     

A morphometric study of normodense and hypodense human eosinophils that are derived in vivo and in vitro.

Hypodense eosinophils were obtained from two patients with the idiopathic hyperosinophilic syndrome (IHES), and hypodense eosinophils were derived by culturing normodense human eosinophils from control donors in the presence of endothelial cells alone, granulocyte/macrophage-colony-stimulating factor (GM-CSF) alone, or GM-CSF and fibroblasts. These eosinophils were examined ultrastructurally and stereologically for alterations in the volume density (Vv) of their electron-dense granules, the Vv of their lucent granules, the Vv of their lipid droplets, the numerical density of their granules with respect to cytoplasm (Nv), and the plasma membrane surface area-to-cell volume ratio (Sv) that might account for their decreased sedimentation density. The hypodense eosinophils that were obtained from the two patients with IHES exhibited a one-third reduction in granule Vv relative to normodense eosinophils from control donors, primarily because of a decrease in granule size. The culture-derived hypodense eosinophils exhibited 10% to 16% decreases in their granule Vv, significant increases in their lucent granules, and a approximately 7.5% decrease in their Sv. Calculation of the cell volume from cross-sectional area measurements showed that the eosinophils that had been cocultured with fibroblasts in the presence of GM-CSF increased their volume by approximately 15%. The eosinophils that had been cocultured with endothelial cells exocytosed some of their granules. In conclusion, a composite of factors including cell swelling, a decrease in the volume of the cytoplasm occupied by granules, and an increase in granule lucency contributes to the hypodense phenotype in vitro, but only cell swelling and hypogranulation are seen in cells from patients with IHES. The latter could reflect the response of 'primed' hypodense eosinophils in vivo to pertinent tissue ligands.     

Human eosinophils are more toxic than neutrophils in antibody-independent killing

Eosinophils (EOSs) are implicated in damaging host tissues in diseases such as asthma and eosinophilic gastroenteritis. In the present study, we assessed the cytotoxicity of human EOSs from peripheral blood of patients with eosinophilia and from peritoneal fluid of patients undergoing continuous peritoneal dialysis and compared them to normal neutrophils. Cytotoxicity was measured by the release of 51chromium from cultured tumor cells and chicken erythrocytes. Both EOSs and neutrophils were separated on discontinuous Percoll gradients with greater than 95% purity. The granulocytes were activated by preincubation in an ice bath with phorbol myristate acetate and washed before incubation with the target cells. The EOSs lysed significantly more tumor cells (K562, Raji, and CEM lines) in an 18-hour assay than did neutrophils, and no significant difference was found between the peritoneal and blood EOSs. The EOSs were also much more efficient than neutrophils in lysing chicken erythrocytes when they were activated by granulocyte-macrophage colony-stimulating factor instead of phorbol myristate acetate. Cytolysis by EOSs is mediated by both oxidative and nonoxidative mechanisms, as indicated by experiments with cells from patients with chronic granulomatous disease. Thus, EOSs are much more cytotoxic than neutrophils and potentially much more damaging to patients with eosinophilia.     

Distinct amplification of the C5a-receptor pathways in normodense and hypodense eosinophils of patients with atopic dermatitis

In patients with atopic dermatitis two different types of blood eosinophils with distinct density can be isolated. The normodense cells represent the huge majority in count, whereas the hypodense eosinophils are characterized by higher effector activity. To understand the altered functional responsiveness of these two cell subtypes, the expression of C5a receptors as well as C5a-induced signal pathways and the production of reactive oxygen metabolites have been analyzed. Chemiluminescence measurements revealed significant higher production of reactive oxygen metabolites in hypodense eosinophils in comparison to normodense cells. However, no difference in the expression level of C5a receptors as well as in the C5a-induced Ca2+-transients between normodense and hypodense eosinophils were found. In contrast, hypodense eosinophils showed a significantly higher actin polymerization response and phosphatidylinositol 4,5 bisphosphate 3-kinase activation after stimulation with C5a than normodense eosinophils. Therefore, normodense and hypodense eosinophils from the blood of patients with atopic dermatitis are characterized by differential amplification of C5a-receptor signal pathways, which might explain the differences in their proinflammatory activity.     

Interleukin-5, interleukin-3 and granulocyte-macrophage colony-stimulating factor prime actin-polymerization in human eosinophils: a study with hypodense and normodense eosinophils from patients with atopic dermatitis

Characteristic features of atopic diseases (AD) are immigration and local activation of eosinophils. Reorganization of the cytoskeleton modulates the function of leukocytes and is a prerequisite for the motility response. In this work, the regulation of actin polymerization has been investigated by flow cytometry using NBD-phallacidin and right angle light scatter measurements in purified eosinophils isolated from patients with atopic dermatitis and normal individuals. Stimulation of eosinophils with chemotaxins such as complement fragment C5a (C5a), CC chemokine RANTES/ CCL5 and platelet activating factor (PAF) induced a reversible polymerization of actin. Normodense eosinophils purified from patients with AD showed a decreased chemotaxin-induced actin response as compared to normodense eosinophils from healthy subjects and hypodense eosinophils from patients. Stimulation of eosinophils with Th2-cytokines such as interleukin-3 (IL-3), interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF) did not exert a significant effect on actin polymerization. However, pretreatment with IL-3, IL-5 or GM-CSF potentiated the chemotaxin-induced actin polymerization and graded the differential responsiveness between normodense and hypodense eosinophils. We demonstrate a different actin responsiveness in eosinophils from atopic patients and healthy subjects which could be overcome by modulating effects of Th2-cytokines.     

Rat mononuclear cells and neutrophils are more effective than eosinophils in antibody-mediated stage-specific killing ofTrichinella spiralis in vitro

The capacity of non-infected rat total, eosinophil-enriched and eosinophil-depleted fractions of peritoneal exudate and bone marrow cells to adhere to and killTrichinella spiralis newborn larvae with immune rat serum has been studied in vitro. The eosinophil-depleted peritoneal exudate cell fraction contained mainly mononuclear cells, whereas the corresponding bone marrow cell fraction consisted of a considerable number of neutrophils. All cell types either originating from the peritoneal cavity or the bone marrow, showed adherence and killing properties to the Trichinella newborn larvae. It was concluded that mononuclear cell and neutrophils are capable of and more effective than eosinophils in stage-specific killing of Trichinella in vitro.     

egc-Encoded superantigens from Staphylococcus aureus are neutralized by human sera much less efficiently than are classical staphylococcal enterotoxins or toxic shock syndrome toxin

PCR was employed to determine the presence of all known superantigen genes (sea, seq, and tst) and of the exotoxin-like gene cluster (set) in 40 Staphylococcus aureus isolates from blood cultures and throat swabs; 28 isolates harbored superantigen genes, five on average, and this strictly correlated with their ability to stimulate T-cell proliferation. In contrast, the set gene cluster was detected in every S. aureus strain, suggesting a nonredundant function for these genes which is different from T-cell activation. No more than 10% of normal human serum samples inhibited the T-cell stimulation elicited by egc-encoded enterotoxins (staphylococcal enterotoxins G, I, M, N, and O), whereas between 32 and 86% neutralized the classical superantigens. Similarly, intravenous human immunoglobulin G preparations inhibited egc-encoded superantigens with 10- to 100-fold-reduced potency compared with the classical enterotoxins. Thus, there are surprisingly large gaps in the capacity of human serum samples to neutralize S. aureus superantigens.     

Functional CD137 receptors are expressed by eosinophils from patients with IgE-mediated allergic responses but not by eosinophils from patients with non-IgE-mediated eosinophilic disorders.

BACKGROUND: CD137 (ILA/4-1BB), a member of the TNF/nerve growth factor receptor superfamily, has previously been suggested to be involved in T-cell activation and differentiation. OBJECTIVE: The aim of this study was to investigate expression and potential function of CD137 in eosinophils. METHODS: Eosinophils were isolated from normal control subjects as well as from patients with bronchial asthma, patients with atopic dermatitis, and patients with idiopathic eosinophilia. CD137 expression was analyzed by RT-PCR and flow cytometry. The in situ expression of CD137 on eosinophils in nasal polyp and skin tissues was analyzed through use of immunohistochemistry. To examine whether CD137 regulates eosinophil death and apoptosis, cells were stimulated with a plate-bound anti-CD137 antibody in the presence or absence of survival cytokines. Cell death was measured by means of an ethidium bromide exclusion test. Apoptosis was determined by analyzing phosphatidylserine surface exposure. RESULTS: Blood and tissue eosinophils from patients with IgE-mediated allergic responses (atopic dermatitis, extrinsic asthma) express CD137. In contrast, eosinophils from normal control individuals and patients with non-IgE-mediated eosinophilic inflammatory responses (intrinsic asthma, idiopathic eosinophilia) express neither detectable levels of mRNA nor protein for CD137. Expression of CD137 in eosinophils was induced in vitro by stimulating the cells with supernatants derived from in vivo- or in vitro-activated T cells, suggesting that a soluble T cell-derived factor might be responsible for the observed phenomenon. Although CD137 expression was associated with increased IgE levels, IL-4 and IL-13 did not induce CD137 gene expression in eosinophils. Activation of CD137 abrogated both GM-CSF-mediated and IL-5-mediated antiapoptosis in CD137-expressing eosinophils but not in CD137-deficient eosinophils. In contrast, the survival effect of IFN-gamma was not affected by anti-CD137 treatment. CONCLUSION: Our data indicate that CD137 activation might limit GM-CSF-mediated and IL-5-mediated antiapoptosis of eosinophils. The absence of this potential anti-inflammatory mechanism might further increase eosinophil numbers at inflammatory sites in patients with intrinsic asthma and patients with idiopathic eosinophilia. The T cell-derived factor that induces CD137 expression in eosinophils remains to be identified.     

32P放射自显影术对家兔经脉循行路线的宏观显示

目的对家兔经脉循行路线进行宏观显示的研究,为细胞水平及超微组织结构的示踪研究奠定基础。方法用穴位测定治疗仪在对应人体的外关、内关、昆仑、太溪经穴处对家兔的经穴定位,经穴位注入Na2H32PO4,用32P整体放射自显影术对家兔经脉循行路线进行宏观显示。结果获得了家兔经脉循行路线整体放射自显影像,影像线性较宽,形状单一且较粗大,影像走向与通过家兔皮肤导电量测定获得的经穴走向相似。结论家兔体表存在与人体相近的具有低阻抗性的经穴,32P放射自显影术可以实现家兔经脉循行路线的宏观显示。     

Retreatment of recurrent cystic craniopharyngioma with chromic phosphorus P 32

A cystic craniopharyngioma in a two-year-old boy recurred six months after surgery and postoperative external-beam radiotherapy. Successful retreatment was accomplished with radioisotope injection of 0.5 mCi of chromic phosphorus P 32 into the intracranial cyst, which delivered approximately 300.00 Gy to the cyst wall. The patient''s symptoms were relieved, and he is without evidence of disease or cystic fluid accumulation four years after intracavitary ³²P irradiation.     

Waves earlier than P3 are more informative in putative subcortical Dementias: A study with mapping and neuropsychological techniques

Summary Thirty subjects (normal controls, patients with putative subcortical dementia and non-demented patient controls) were studied using advanced neurophysiological (16 scalp-electrode positions, computer-assisted brain electrical activity mapping, auditory oddball paradigm) and neuropsychological techniques. Our study suggests that waves earlier than P₃ (N₁, P₂ and N₂) are all correlated with global measures of cognitive functions. They are, however, differentially correlated with specific measures of cognitive functions, N₁ and P₂ with mental speed and N₂ with short-term memory. The abnormalities of these waves (earlier than P₃) may be an electrophysiologic marker of dementia in patients with putative subcortical states.     

Immunoprecipitation of antigen-associated [32P]-labelled nucleic acids from Crohn's disease mesenteric lymph nodes

The methods of immunoselection and electrophoretic analysis of [32P]-labelled nucleic acids have been applied to the problem of defining Crohn's disease (CD) specific antigen associated DNA or RNA, with the intention of identifying a presumptive aetiological microbial agent. Mesenteric lymph node derived cells from CD and control gastrointestinal disease cases were cultured in vitro with [32P] orthophosphate after mitogenic stimulation with phytohaemaglutinin and pokeweed mitogen. Total cell lysates were immunoprecipitated with CD and control serum IgG fractions and immune complexes recovered with pansorbin. Antigen associated [32P]-labelled nucleic acids were phenol/chloroform extracted and analysed by electrophoresis on polyacrylamide and agarose gels. No immunoprecipitated nucleic acid specific to CD tissues could be detected and no differences in antigen recognition between CD and control serum IgG were observed. No evidence was obtained for nucleic acid containing antigens either of the autoimmune type or of possible viral or microbial origin in CD mesenteric lymph nodes.     

On a much higher than reported incidence of anti-c in R1R1 patients with anti-E

A previous study involving tube IATs, untreated RBCs, and a low ionic-strength additive reagent revealed that approximately one-third of R(1)R(1) patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c in such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies and transfusion records of 82 R(1)R(1) patients with anti-E were reviewed. Serologic test methods included a LISS wash solution for tube IATs (15 min at 37 degrees C, anti-IgG), ficin-tube IATs (30 min at 37 degrees C, anti-IgG + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated RBCs were used, 21 additional patients with anti-E were found to have anti-c. In samples from 26 R(1)R(1) patients with anti-E, anti-c was not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E- RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be without anti-c, all subsequently performed crossmatches with E-, c-untested RBCs were compatible. The incidence of anti-c in R(1)R(1) patients with anti-E in this study was 32 of 82 (39%) with untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. The latter is significantly higher than the 32 percent incidence previously reported (p = 0.0001). Accordingly, all patients at our facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. The data from this study strongly support the selection of R(1)R(1) RBCs for all c- patients with anti-E.     

Dot-blot procedure with [32P]DNA probes for the sensitive detection of avocado sunblotch and other viroids in plants

Avocado sunblotch viroid (ASBV) has been detected down to a level of about 20 pg per gram fresh weight of leaves by the use of a dot-blot hybridization procedure and partially purified nucleic acid extracts. Three [32P]DNA probes were compared, two prepared from full-length ASBV clones in the single-strand M13mp93 vector and the other by primer extension on purified ASBV. All three probes gave the same sensitivity of detection of ASBV. The methods developed have also been used successfully for the routine detection of potato spindle tuber viroid, citrus exocortis viroid, and coconut cadang cadang viroid.     

Immunoprecipitation of antigen-associated [32P]-labelled nucleic acids from Crohn's disease mesenteric lymph nodes.

The methods of immunoselection and electrophoretic analysis of [32P]-labelled nucleic acids have been applied to the problem of defining Crohn''s disease (CD) specific antigen associated DNA or RNA, with the intention of identifying a presumptive aetiological microbial agent. Mesenteric lymph node derived cells from CD and control gastrointestinal disease cases were cultured in vitro with [32P] orthophosphate after mitogenic stimulation with phytohaemaglutinin and pokeweed mitogen. Total cell lysates were immunoprecipitated with CD and control serum IgG fractions and immune complexes recovered with pansorbin. Antigen associated [32P]-labelled nucleic acids were phenol/chloroform extracted and analysed by electrophoresis on polyacrylamide and agarose gels. No immunoprecipitated nucleic acid specific to CD tissues could be detected and no differences in antigen recognition between CD and control serum IgG were observed. No evidence was obtained for nucleic acid containing antigens either of the autoimmune type or of possible viral or microbial origin in CD mesenteric lymph nodes.