Host Plasma Proteins Research Articles

Nonvalvular cardiovascular device-related infections.

More than a century ago, Osler took numerous syndrome descriptions of cardiac valvular infection that were incomplete and confusing and categorized them into the cardiovascular infections known as infective endocarditis. Because he was both a clinician and a pathologist, he was able to provide a meaningful outline of this complex disease. Technical advances have allowed us to better subcategorize infective endocarditis on the basis of microbiological etiology. More recently, the syndromes of infective endocarditis and endarteritis have been expanded to include infections involving a variety of cardiovascular prostheses and devices that are used to replace or assist damaged or dysfunctional tissues (Table 1). Taken together, infections of these novel intracardiac, arterial, and venous devices are frequently seen in medical centers throughout the developed world. In response, the American Heart Association’s Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease wrote this review to assist and educate clinicians who care for an increasing number of patients with nonvalvular cardiovascular device–related infections. Because timely guidelines1,2 exist that address the prevention and management of intravascular catheter–related infections, these device-related infections are not discussed in the present Statement. View this table: TABLE 1. Nonvalvular Cardiovascular Device–Related Infections This review is divided into two broad sections. The first section examines general principles for the evaluation and management of infection that apply to all nonvalvular cardiovascular devices. Despite the marked variability in composition, structure, function, and frequency of infection among the various types of nonvalvular cardiovascular devices reviewed in this article, there are several areas of commonality for infection of these devices. These include clinical manifestations, microbiology, pathogenesis, diagnosis, treatment, and prevention. The second section addresses each device and describes unique clinical features of infection. Each device is placed into one of 3 categories—intracardiac, arterial, or venous—for discussion. ### Clinical Manifestations The specific signs and symptoms associated with an infection of a …

Heat elution chromatography of immunoglobulins

A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We present a new and simplified method to obtain pure antibody based on the special thermal properties of the streptococcal M proteins, a family of cell–surface exposed coiled-coil molecules which bind different sets of host plasma proteins. The coiled-coil structure is already destabilized at low temperatures and the M proteins unfold reversibly, usually below 40 °C. We demonstrate the use of this property to purify immunoglobulin G from rabbit serum with protein H from the AP1 strain of Streptococcus pyogenes. Recombinant protein H is linked to nickel–agarose via a C-terminal histidine tag. After mixing with rabbit serum and washing at room temperature, pure IgG can be eluted from the gel with a moderately heated buffer. In this case, protein H has been used to purify rabbit IgG, but the principle should be applicable to other M protein–ligand pairs.

Parasitism of Lacanobia oleracea (lepidoptera) by the ectoparasitic wasp, Eulophus pennicornis, disrupts the cytoskeleton of host haemocytes and suppresses encapsulation in vivo.

Parasitism of Lacanobia oleracea larvae by the ectoparasitic wasp Eulophus pennicornis suppressed host haemocyte-mediated encapsulation of Sephadex DEAE A-25 beads in vivo. Beads dissected out of parasitized larvae had fewer haemocytes associated with them. Moreover, those haemocytes that were associated with the beads tended to retain a rounded configuration and rarely flattened. Similar results were obtained using in vitro encapsulation assays. SDS PAGE indicated that for parasitized and PBS injected larvae, there were some differences in the plasma proteins that bound to Sephadex DEAE A-25 beads, suggesting that parasitism-mediated changes to host plasma proteins might contribute to the differences in the encapsulation response occurring in these larvae. However, in vitro encapsulation assays using beads that had been pre-incubated in plasma from parasitized and unparasitized larvae, demonstrated that major differences in the extent of encapsulation did not occur. These results, plus in vitro haemocyte attachment and spreading assays, suggest that parasitism-mediated suppression of encapsulation is primarily due to reductions in the ability of host haemocytes to attach to (i.e., recognize) and flatten over non-self surfaces and other haemocytes. This proposal is corroborated by staining of actin in the haemocyte cytoskeleton by FITC-labelled phalloidin, which indicated that parasitism disrupts the formation of stress fibers and focal adhesions in plasmatocytes. By contrast, experimental injection of adult female wasp venom into unparasitized L. oleracea larvae had no significant effect on in vivo encapsulation responses or the haemocyte cytoskeleton. Arch. Insect Biochem. Physiol. 49:108-124, 2002. Published 2002 Wiley-Liss, Inc.

Host hemolymph proteins and protein digestion in larval Habrobracon hebetor (Hymenoptera: Braconidae)

Host plasma proteins and protein digestion in larval parasitoids were studied during trophic interactions of the ectoparasitoid Habrobracon hebetor Say (Hymenoptera: Braconidae), with a host, larvae of the Indianmeal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae). We could detect no apparent differences in host hemolymph protein patterns up to 72 h after paralysation and/or parasitization by H. hebetor. A 190 kDa putative apolipophorin I present in host hemolymph could not be detected in the midguts of feeding H. hebetor larvae indicating that it is rapidly digested. The major 60 kDa storage proteins (putative hexamerins) in host hemolymph were detected in the parasitoid midgut and were completely digested 24 h after cessation of feeding and the beginning of cocoon formation. Host hemolymph had a pH of about 6.4. The pH optima of the midgut proteinases in the larval parasitoid were in the alkaline region, but midgut fluid in feeding parasitoid larvae was about pH 6.8. Based on enzyme activity against selected artificial proteinase substrates including azocasein, N-α-benzoyl-L-Arg p-nitroanilide (BA pNA), succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPF pNA), succinyl-Ala-Ala-Pro-Leu p-nitroanilide (SAAPL pNA), and inhibition by selected proteinase inhibitors, serine proteinases appear to be the predominant class of enzymes involved in protein digestion in the midguts of H. hebetor. There is also an active aminopeptidase (L pNA) associated with the microsomal fraction of midgut preparations. There was no evidence for preoral digestion or ingestion of proteinases from host hemolymph by the parasitoid larva. There was a very active BA pNAase in the soluble fraction of midgut extracts. This activity increased on a per midgut basis up to 24 h after the beginning of cocoon formation but decreased rapidly by 48 h. Two major (P1 and P3) and several minor proteinases were detected in midgut extracts of H. hebetor analysed with gelatin zymograms. The apparent molecular mass of P1 varied from 95 to 49 kDa depending on protein loading. P3 had an apparent molecular mass of 39 kDa that was independent of protein loading. In summary, electrophoretic evidence indicates that host hemolymph protein patterns do not change significantly for at least 72 h after paralysation by H. hebetor. The role, if any, of envenomization in preventing breakdown of hemolymph proteins during this time remains to be determined. Because the predominant host hemolymph proteins, a putative apolipophorin I and the putative hexamerins, are readily digested by the serine proteinases present in the midguts of this parasitoid larva, these or similar proteins would provide an easily digested source of dietary amino acids that could be used for development of artificial diets for this beneficial insect.

Parasitism of Lacanobia oleracea (Lepidoptera, noctuidae) by the ectoparasitic wasp Eulophus pennicornis, results in the appearance of a 27 kDa parasitism-specific protein in host plasma

Little is known about the effects of ectoparasitoids and their secretions on the plasma protein profiles of their insect hosts. To address this deficit, a study has been made of the interactions between an ectoparasitic wasp, Eulophus pennicornis, and its host, the tomato moth, Lacanobia oleracea. In particular, the quantitative and qualitative effects of parasitism or the experimental injection of wasp venom on host plasma proteins were investigated. Results demonstrated that both treatments caused an initial increase in L. oleracea total plasma protein concentration up to day 5 of treatment, but whereas the protein concentration remained high in the experimentally envenomated group, a decrease towards day 8 occurred in parasitized insects. Parasitism was also associated with the appearance of a protein with an estimated molecular weight of 27 kDa. This protein first appeared on day 3 after parasitization and its levels subsequently increased. The protein was not detected in any of the unparasitized larvae (including all the various control groups) or in experimentally envenomated L. oleracea larvae. In addition, the appearance of this protein was not a non-specific result of nutritional deprivation, nor was it a general injury, stress, or infection induced protein. Its appearance was strictly associated with parasitism of L. oleracea by E. pennicornis and thus, it may be described as a parasitism-specific protein (PSP). The PSP has been partially purified using whole gel elution. Gel filtration and SDS PAGE indicated that it has a native molecular weight of 27 kDa and that it does not appear to aggregate to produce higher molecular weight molecules, nor dissociate into lower molecular weight subunits held together by disulphide or covalent bonds. The precise site of synthesis of the 27 kDa PSP is not yet known but some evidence leads us to speculate that it may be synthesised by the feeding E. pennicornis larvae and introduced into their host. This possibility is discussed in relation to previous work detailing the effects of parasitism on L. oleracea haemocyte morphology, function and viability, and the effects of endoparasitoids on host plasma proteins.

Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins.

Adherence of Candida albicans to host tissues is a necessary step for maintenance of its commensal status and is likely a necessary step in the pathogenesis of candidiasis. The extracellular matrix (ECM) proteins are some of the host tissue and plasma proteins to which C. albicans adheres through adhesins located on the fungal cell surface. To isolate genes encoding ECM adhesins, an assay was developed based on the ability of yeast cells to adhere to magnetic beads coated with the ECM protein fibronectin, type IV collagen, or laminin. A C. albicans genomic library was constructed by cloning XbaI-partially-digested and size-selected fragments into pAUR112, an Escherichia coli-yeast low-copy-number shuttle vector. The C. albicans library was transformed into Saccharomyces cerevisiae YPH 499, and clones capable of adherence were selected by using ECM protein-coated magnetic beads. A plasmid containing an approximately 8-kb insert was isolated from 29 adherent clones. These clones exhibited adherence to all ECM protein-coated magnetic beads and to human buccal epithelial cells. The ALA1 gene (for agglutinin-like adhesin) was localized by subcloning it into a 5-kb XbaI fragment which retained the adherence phenotype in both orientations. The complete DNA sequence of the 5-kb insert was determined, and an open reading frame (ORF) encoding 1,419 amino acid residues was identified. Deletions from the 5' and 3' ends extending into the DNA sequence encoding the 1,419-amino-acid ORF product inactivated the adherence phenotype, suggesting that it is the coding region of the ALA1 gene. A database search identified ALA1 to be similar to the C. albicans ALS1 (for agglutinin-like sequence 1) protein and the S. cerevisiae agglutinin protein (AG alpha1), although the homology at the primary amino acid sequence level is limited to the first half of each of these proteins. ALA1 contains a central domain of six tandem repeats of 36 amino acids. We discuss the significance of various predicted ALA1 structural motifs and their relationships to function in the adherence process.

Receptor-mediated endocytosis in the bloodstream form of Trypanosoma brucei.

The uptake of various host plasma proteins by the bloodstream form of Trypanosoma brucei was studied both biochemically, using radiolabeled proteins, and with the electron microscope, using colloidal gold particles as molecular tracers onto which plasma proteins had been adsorbed. Total plasma proteins and serum albumin were taken up by a mechanism of fluid endocytosis with low clearance (0.1 microliter [mg cell protein]-1 h-1), while low-density lipoprotein (LDL) and transferrin were taken up by a receptor-mediated process with a clearance of two to three orders of magnitude higher than that of serum albumin. Binding prior to uptake of LDL and transferrin was saturable, depended on the presence of Ca2+, and the labeled ligand could be displaced by the homologous but not by heterologous protein. Binding of gold-labeled proteins was seen only to the membrane of the flagellar pocket and not elsewhere on the plasma membrane. After 1 h of incubation at 30 degrees C with gold-labeled LDL and transferrin, labeled cellular structures represented respectively half and one-third of the total volume of all single-membrane bounded endocytotic and electron-dense vacuoles within the cell.

Acquisition of alpha 1-Antitrypsin by a pathogenic strain of Trichomonas vaginalis.

The interaction of alpha 1-Antitrypsin, the major serine protease inhibitor in plasma, with pathogenic Trichomonas vaginalis and the acquisition by trichomonads of this host protein from normal human plasma were investigated. alpha 1-Antitrypsin acquired by intact parasites could not be removed by repeated washings in phosphate-buffered saline. Saturation kinetics were observed after incubation of glutaraldehyde-fixed organisms with 125I-labeled alpha 1-antitrypsin. Evidence suggesting that specific parasite membrane sites were responsible for trichomonal acquisition of alpha 1-antitrypsin was obtained through competitive binding experiments using purified preparations of homologous versus heterologous plasma proteins. No evidence of degradation of bound antitrypsin by live parasites was observed. The avid binding of alpha 1-antitrypsin by pathogenic T. vaginalis after incubation in normal human plasma was demonstrated by using sensitive electrophoretic and immunodetection techniques. Radioimmunoprecipitation of intrinsically labeled, detergent-solubilized extracts of T. vaginalis incubated with monospecific antisera against alpha 1-antitrypsin and other human plasma proteins revealed the inability of parasites to biosynthesize any substance cross-reactive with host plasma proteins. Finally, T. vaginalis organisms pretreated with alpha 1-antitrypsin inhibited trypsin caseinase activity in an in vitro assay. The implications of these observations are discussed.

Host plasma proteins on the surface of pathogenic Trichomonas vaginalis.

Sodium dodecyl sulfate-gel electrophoresis and fluorography and fluorography technology revealed that pathogenic Trichomonas vaginalis was able to acquire numerous loosely associated plasma proteins during incubation in normal human plasma. These proteins were readily removed by repeated washing of the parasite in phosphate-buffered saline. Plasma proteins avidly bound to the surface of T. vaginalis were also detected using a highly sensitive and specific agglutination assay with protein A-bearing Staphylococcus aureus pretreated with monospecific antiserum directed against individual human serum proteins. These avidly associated plasma proteins could not be removed by repeated washing in phosphate-buffered saline or by treatment of washed, live organisms with surface-modifying reagents such as trypsin and periodate. A combined radioimmunoprecipitation-gel electrophoresis-fluorography methodology indicated that parasite biosynthesis of hostlike macromolecules was not responsible for the observed agglutination and reinforced the idea of trichosomal acquisition of plasma components. Finally, incubation of trichomonads with plasma in various buffers at different pH values did not alter the agglutination patterns. These and other data suggest that specific membrane sites trichomonal binding of host proteins. The biological significance of our results is discussed.

Comparative Immunological Analysis of Host Plasma Proteins Bound to Bloodstream Forms of Trypanosoma brucei Subspecies

The presence, location, host specificity, identity, and quantity of rat plasma proteins bound to bloodstream forms of Trypanosoma brucei subsp. brucei, T. brucei subsp. rhodesiense, and T. brucei subsp. gambiense were determined by a quantitative indirect fluorescent-antibody method and gel immunoassays. Fluorescence differences between trypanosomes obtained from rats and mice and treated with antiserum to normal rat plasma indicated that most, if not all, of the bound plasma proteins were host specific. Removal of plasma proteins by trypsinization of parasites provided evidence for their attachment to the surface of the parasite. The accreted proteins were found to be host albumin, immunoglobulin G (IgG), and complement (C3). The same quantities of these three plasma proteins were present on T. brucei subspecies collected from normal rats at 2 days postinfection, during low or peak parasitemias, or from cortisone-treated rats. IgM could only be detected on parasites collected from normal rats at peak parasitemia. With the aid of rocket immunoelectrophoresis, host albumin and IgG were found to account for 0.2 and 0.05%, respectively, of the total soluble proteins of the bloodstream forms. It was concluded from this study that: (i) host plasma proteins were bound to parasites early in the infection, suggesting a mechanism of adaptation to the mammalian host; (ii) the surface-bound IgG was not the result of a specific immune response against the parasites but might be the cause of C3 attachment; (iii) among the bloodstream forms of the three T. brucei subspecies, there were no differences in amounts of surface-bound albumin, IgG, or C3. A comparison between the present data dealing with T. brucei subspecies, on the one hand, and the previously published results concerning T. congolense, on the other, revealed significant differences in the amounts of the host plasma proteins attached to these hemoflagellates.

Fluorescent Antibody Analysis of Host Plasma Components on Bloodstream Forms of African Pathogenic Trypanosomes. I. Host Specificity and Time of Accretion in Trypanosoma congolense

The presence, host specificity, and time of accretion of rat plasma components on the surface of Trypanosoma congolense were determined. Parasites collected at peak parasitemia (6to 10-day rat infections) were separated from blood cells on a DEAE-cellulose column and exogenous plasma was removed by washing. For immunoelectrophoretic analysis, the trypanosomes were lysed, the lysate centrifuged, and the soluble antigen reacted with rabbit antiserum developed against normal rat plasma. At least two antigen-antibody complexes were observed. To determine the location, host specificity, and time of appearance of the antigens shared by the host and parasite, a quantitative indirect fluorescent antibody method was used. The washed trypanosomes were fixed in 2.5% or 10% formalin before being subjected to the sandwich method. Formalin at a 2.5% concentration was the most suitable for the preservation of antigens. It was established by titrations that the largest difference in the fluorescence levels of the experimental and experimental control preparations occurred when the nonconjugated (first layer) rabbit antisera were used at 1:16 dilutions, and the fluorescein isothiocyanateconjugated anti-rabbit IgG serum (second layer) at a 1:40 dilution. Fluorescence differences between ratand mouse-derived trypanosomes stained with anti-rat plasma serum indicated that most, if not all, of the plasma components were host-specific. In vivo and in vitro experiments were performed to determine the length of time needed for appearance of new-host plasma components on the trypanosome surface. The rat-derived bloodstream forms when transferred to mice for 48 hr or incubated for 5 hr in normal mouse plasma emitted fluorescence quantitatively identical to that of trypanosomes grown in mice by serial passages. Incubation of mouse-derived trypanosomes in normal mouse plasma for 5 hr did not change their fluorescence measurements, but such trypanosomes incubated for 3 to 5 hr in normal rat plasma had fluorescence levels similar to those of rat-derived trypanosomes. The speed of acquisition and host specificity of the surface-bound antigens suggested that they were host plasma components accreted on the parasites. The occurrence of host plasma proteins on the surfaces of trypanosomes was suggested originally by Desowitz and Watson (1953) with regard to a rodent-adapted strain of Trypanosoma vivax. Subsequently, antigens common to trypanosomes and their hosts were reported from bloodstream forms of stercorarian and salivarian species on the bases of the results obtained by agglutination, double diffusion, immunoelectrophoresis, and direct fluorescent antibody methods. Among Stercoraria, such antigens were observed in Trypanosoma lewisi by D'Alesandro (1972) and Dwyer (1976). Among Salivaria, they were found in a mouse-adapted T. vivax (Ketteridge, 1970), Received for publication 8 December 1976. * This investigation was supported by Research Grant AI 00742-21, from NIAID, U.S. Public Health Service. t Predoctoral Trainee, supported by Training Grant 5 T01 AI 00226-13, from NIAID, U.S. Public Health Service. rat-adapted Trypanosoma brucei gambiense (Seed, 1974), and Trypanosoma brucei brucei (Vickerman, 1972; Le Ray, 1975). The present study was undertaken to ascertain if antigens resembling those of the host plasma occur on Trypanosoma (Nannomonas) congolense Broden. Since such host-parasite shared components were found, in vivo and in vitro experiments were designed to determine the length of time needed for the appearance of new host plasma components on the surface of the parasites. MATERIALS AND METHODS