Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmiter of the trematode Schistosoma mansoni in Brazil, were examined by ligth and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite

Interferon affects nuclear proteins in cells of clinically sensitive chronic myelogenous leukemia patients.

Cytoplasmic protein extracts from chronic myelogenous leukemia (CML) cells contained an activity that altered the electrophoretic mobility of complexes formed between nuclear proteins and the transcriptional enhancers of interferon (IFN)-inducible genes. Exposure of CML cells to IFN-alpha diminished the effect of the CML cytoplasmic proteins on these nuclear protein-DNA complexes. The presence of clinical responsiveness to IFN-alpha correlated with the sensitivity to the IFN-induced change in the electrophoretic mobility of nuclear protein-DNA complexes. These data suggest that the action of IFN-alpha in CML may be linked to a pathway that can result in posttranslational modification of nuclear proteins.

Immunocytochemical evidence of vertebrate bioactive peptide-like molecules in the immuno cell types of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata).

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.

In situ detection of basic fibroblast growth factor by highly specific antibodies.

Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors.

A monoclonal antibody to a megakaryoblast-like cell line detects a protein found in blood cells and in the epithelial cell lining of various rat tissues.

Megakaryoblasts of bone marrow differentiate into megakaryocytes that in turn are the source of blood platelets. We have raised monoclonal antibodies to a megakaryoblast-like cell line derived from rat bone marrow (RPM cells). One antibody (Mab 213) and the corresponding antigen has been characterized by Western blotting and immunohistochemistry. Biosynthetic labeling with [35S]methionine showed that this antigen is synthesized by the RPM cells. In Western blots the antibody recognized proteins of about 90 kDa and 160 kDa in Triton extracts of RPM cells, whereas it recognized proteins of about 160 kDa and 200 kDa in Triton extracts of rat platelets and one of about 200 kDa in Triton extracts of various rat tissues (kidney, lung, intestine, and heart). By immunohistochemistry, the antigen was localized to the apical part of the epithelium lining certain parts of kidney tubuli, bronchi and large intestine.

Immunocytochemical localization of a calcium-binding phosphoprotein in hemocytes of heterodont bivalves.

Calcium-binding phosphoprotein particles are the most abundant extracellular proteins in the hemolymph of heterodont bivalves, and granular hemocytes are the most abundant cells in the same fluid. In this study, the hemocytes of Rangia cuneata were examined ultrastructurally and probed with anti-phosphoprotein IgG to demonstrate that the granulocytes are a probable source of the hemolymph phosphoprotein. The granulocyte cytoplasm is laden with large vesicles containing an amorphous homogenous matrix and variable numbers of electron-dense particles; the latter are ultrastructurally similar to the extracellular phosphoprotein. The vesicle particles and matrix are related forms of the hemolymph phosphoprotein as evidenced by heavy gold labeling when Lowicryl sections were sequentially treated with rabbit-anti-phosphoprotein IgG and colloidal gold-anti-rabbit IgG. The vesicles may be the loci for posttranslational phosphorylation and eventual secretion of the calcium-binding phosphoprotein, or alternatively the vesicles may be digestive structures which degrade internalized phosphoprotein.

Glycosphingolipids of Echinococcus multilocularis metacestodes.

Neutral and acid glycosphingolipids of Echinococcus multilocularis metacestodes that were obtained after intraperitoneal infection of Meriones unguiculatus have been analyzed by thin layer chromatography. Neutral and acid glycosphingolipids accounted for 95% and 5% of total glycosphingolipids, respectively. 12 different fractions were observed in the neutral glycosphingolipids extracts of the parasite. The most important was a monohexosylceramide fraction accounting for 56.4% of neutral glycosphingolipids. 9 different fractions were detected in gangliosides (acid glycosphingolipids). The fact that these glycosphingolipids were specific to the parasite was established by the analysis of different cell populations of the host. Glycosphingolipids were purified from control and parasite-infected gerbil blood cells as well as from peritoneal exudate cells of healthy gerbils after a non-specific immunostimulation. The chromatograms obtained with these extracts were totally different from the parasite. In addition, parasitosis was found to have no effect on the host blood cell glycosphingolipids.

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma.

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.

Immunodetection of haemocyte subpopulations by N-acetylmuramic acid antibody in Planorbarius corneus (L.) (Gastropoda, Pulmonata).

The cell subpopulations in the haemolymph of Planorbarius corneus were distinguished by means of flow cytometry. An antibody against N-acetylmuramic acid was prepared and used as a cellular marker to recognize the cell types forming the subpopulations. The spreading haemocytes showed a positive reaction for anti-N-acetylmuramic acid; round haemocytes gave a negative reaction.

Using clinical data to predict abnormal serum electrolytes and blood cell profiles.

OBJECTIVE: To identify clinical predictors of five abnormalities on the serum electrolyte panel and two abnormalities on the blood cell profile, to study which data elements carried predictive information, and to measure the predictive accuracy and stability of the resulting predictive equations. DESIGN: Prospective data collection from a computerized medical database supplemented by data entered by physicians who ordered outpatient tests into microcomputers. Equations were derived during an eight-month period and later validated twice in the same setting. SETTING: Academic primary care practice affiliated with a county hospital. PATIENTS AND PARTICIPANTS: Patients were mostly black women; physicians were full-time academic general internists and medical residents. MEASUREMENTS AND MAIN RESULTS: There were 6,570 electrolyte and blood cell profile panels ordered during the equation derivation period. The mean receiver operating characteristic (ROC) curve area for the seven equations was 0.849. For the 4,977 tests ordered during ten months of prospective validation, the mean ROC curve area was only 3% less. For three equations, ROC curve areas were lower for patients with unscheduled visits than for those with scheduled visits (p less than 0.05). Except for two equations involving abnormalities with very low prevalences, the equations were also well calibrated. Prior results for the abnormality being considered were the strongest predictors, followed by other laboratory results, diagnoses, and the physicians' estimate of the probability that the test would be abnormal. CONCLUSIONS: Clinical data can accurately predict abnormal results of common outpatient laboratory tests. Computers can help find the necessary data and produce estimates of risk.

Studies on the defect which causes absence of decay accelerating factor (DAF) from the peripheral blood cells of an individual with the Inab phenotype.

1. We have studied the peripheral blood cells of an individual with the Inab phenotype who is deficient in decay accelerating factor (DAF). 2. In contrast with the situation in paroxysmal nocturnal haemoglobinuria, membranes from peripheral blood cells of the Inab phenotype individual lack DAF, but retain the other glycosylphosphatidylinositol-linked proteins acetylcholinesterase and LFA-3. 3. Unlike normal Epstein-Barr-virus-transformed lymphoblastoid cell lines (EBV-LCL), DAF was not expressed on EBV-LCL derived from peripheral blood lymphocytes of the Inab individual. 4. No differences in the DAF gene of normal and Inab phenotype individuals could be detected by Southern blotting studies. 5. EBV-LCL derived from the Inab individual had a gross reduction in the level of DAF mRNA compared with normal EBV-LCL. 6. Our results suggest that the DAF gene in the Inab phenotype contains a mutation which affects the transcription or processing of DAF mRNA.

Distribution of tunichrome and vanadium in sea squirt blood cells sorted by flow cytometry.

Specialized blood cells of many tunicates accumulate high concentrations of vanadium and phenolic peptide pigments called tunichromes (TC). In order to determine whether V and TC reside in the same cells, Ascidia nigra and Ascidia ceratodes blood cell subpopulations were isolated by fluorescence-activated cell sorting (flow cytometry) and chemically analyzed. V was found in the spherical, green/grey signet ring cells, and to a lesser degree in the mulberry-shaped, yellow/green morula cells (MRs), whereas free TC was detected mainly in MRs.

[A method of determining the magnitude of the intracellular reserve of heparin in the blood]. / Sposob opredeleniia velichiny rezerva vnutrikletochnogo geparina krovi.

The new method for assessment of the function of the blood anticoagulation system cellular component is based on the detection of the intracellular heparin amount that can be released into the blood stream. The technique involves the recording of changes in the blood plasma heparin levels after addition of lactobacterin, a nonspecific cell stimulant, to whole blood. The informative value of this method for the assessment of the blood anticoagulation system status is illustrated with examples.

Sugar specific cellular lectins of Phallusia mamillata hemocytes: purification, characterization and evidence for cell surface localization.

Cellular lectins (CLs) of Phallusia mamillata were demonstrated in protein preparations obtained by salt fractionation from hemocytes sonicated in a suitable medium. Since the lectins from the precipitated fraction bind sugars containing D-galactosyl groups, they were purified by affinity chromatography on Sepharose. SDS-PAGE under reducing conditions showed that CLs are formed of two components of apparent MWs approximately 36,900 and 35,090 and thus differ from serum lectins (SLs) (MW about 62,200). The "shrinkage" observed when SLs were examined under nonreducing conditions suggest the presence of intrachain disulphide bonds which can affect the molecular structure of the SLs. CL-SL differences were also revealed by the nonidentity reaction of the immuno-precipitate in immunodiffusion using an anti-SL immune serum. The capacity of hemocytes to form rosettes or clumps with erythrocytes demonstrated that they possess alpha-lactose specific CLs on their surfaces.

Intracellular sodium and potassium and their relationship to blood pressure and body size

In the present study twenty-seven hypertensive patients, thirty-five normotensives without familial hypertension and twelve normotensives with familial hypertension were studied cross-sectoinally with regard to their age, sex, body mass index and - most important of all - the electrolyte (sodium and potassium) composition of their red and white blood cells. The major aim of this study was to find out if there were differences between the above mentioned subject groups regarding the electrolyte composition (sodium and potassium) of their blood cells. Another aim of this study was to characterize, through multiple regression analysis, the relationship between blood pressure, body mass index, and cell sodium and potasssium. Higher RBC-Na and WBC-Na were observed in essential hypertensives and normotensives with familial hypertension versus normotensive controls without familial hypertension. Normotensives with familial hypertension had WBC-Na and RBC-Na that were not significantly different from those in essential hypertensives. RBC-K was not significantly different between normotensives with familial hypertension, normotensives without familial hypertension and essential hypertensives. WBC-K was not significantly different between normotensives with familial hypertension and essential hypertensives. Normotensives with familial hypertension had significantly higher WBC-K than normotensives without familial hypertension and essential hypertensives. Correlations that were significant in the combined normotensive-hypertensive group include: (i) The direct relationship between blood pressure (systolic, diastolic and mean) and RBC-Na and also WBC-Na. (ii) The inverse relationship between blood pressure (diastolic and mean) and RBC-K and WBC-K. (iii) The direct relationship between body mass index and WBC-Na and WBC-K. Correlations that were significant in the essential hypertensive group include: (i) The inverse relationship between blood pressure (diastolic and mean) and RBC-K and also WBC-K. (ii) The direct relationship between body mass index and WBC-Na. Most of these results are compatible with the hypothesis that sodium is involved in the pathogenesis of essential hypertension (AU)

Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure.

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.

The host-parasite interface in molluscan schistosomiasis: biotin as a probe for sporocyst and hemocyte surface peptides.

Hemocytes of Biomphalaria glabrata mediate the internal defensive response which, in resistant snail strains, kills sporocysts of Schistosoma mansoni. Lacking a gut, the sporocyst has only its tegument to interact with the host milieu (hemolymph). We have, therefore, focused our study on the surface-exposed proteins of hemocytes and sporocyst tegument. Using gentle biotinylation of living systems, labelled proteins were studied after SDS-PAGE electrophoresis and transfer to nitrocellulose. Results validate the utility of surface biotinylation in studies of host and parasite interfaces. A low diversity characterizes hemocyte surfaces and strain-specific differences are not in evidence. Hemocyte surfaces differ distinctly from the plasma in which these cells reside. In contrast, sporocyst surfaces expose a wide variety of peptides. These are remarkably stable even when sporocysts procured in snail plasma-free media are exposed to plasma. Thus, antigenic differences seen previously when Western immunoblotting was used to study sporocyst surfaces appear to be manifestations of minor changes in the exposed peptides or changes not detectable with this methodology. Hemoglobin, acquired by sporocysts from snail plasma, is processed and disappears from the surface during an overnight chase in culture medium.