Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Ablastin: Control of Trypanosoma musculi infections in mice

Trypanosoma musculi infections in CBA mice consist of a phase of increasing parasitemia during which dividing forms of the parasite are present in the blood, followed by a period when only nondividing trypomastigotes are seen. A second crisis terminates the blood infection and leaves the host immune, but small numbers of trypanosomes, including multiplicative forms, persist in the kidneys for many months. Studies were made involving infections in T-lymphocyte deprived mice, the effects of passive transfer of serum and cells, measurement of DNA synthesis by the parasite, serological responses, and in vitro effects of serum on the trypanosomes. These indicated that the initial check on the increase in blood parasitemia is due in part to two humoral factors. One of these has a trypanocidal effect (this is thought to be an IgM antibody) while the other, which may be an IgG antibody, is the ablastin that inhibits further reproduction by the parasite. Both trypanocidal and ablastic effects were demonstrable in the serum of immune mice yet the parasite was still able to survive and multiply in the kidneys.     

A medium for the continuous cultivation of bloodstream forms of Trypanosoma lewisi at 37 C

Trypanosoma lewisi has been maintained continuously at 37 C for more than 2 yr in Iscove's modified Dulbecco's medium with 10% fetal calf serum and a feeder layer of rat fibroblasts. In this medium the continuously reproducing hematozoic culture forms resemble bloodstream forms of T. lewisi in that they appear morphologically similar in Giemsa-stained preparations examined by light microscopy and have a surface coat that is absent in culture forms grown at ambient temperatures, when examined by electron microscopy. To determine whether these hematozoic culture forms also are similar functionally to bloodstream forms, comparative tests of the 2 forms were made of infectivity for the natural rat host, growth in vitro in the described culture medium, sensitivity to inhibition of reproduction by the rat antibody ablastin, and agglutinability by the 2 trypanocidal antibodies produced during a natural course of infection in the rat. Initially, differences between the 2 forms were minor, but after 16 mo in vitro greater differences began to emerge. Most marked was a reduction in infectivity by 22 mo, although sensitivity to ablastin, the single most important characteristic of bloodstream forms of T. lewisi, was still appreciable at this time. Nevertheless, despite this limitation, the culture system described supports hematozoic culture forms of T. lewisi for a considerably longer time than has been reported thus far.     

Immunosuppression and ablastin

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi. Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species.Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.     

Ablastin in Trypanosoma lewisi and related phenomena in other species of trypanosomes.

The value of comparing processes in related species is emphasized; by this approach it is possible to consider the ablastin phenomenon, not as a unique process, but as having metamorphosis evolutionary affinity with conversion factors in other species of trypanosomes.     

The ablastin phenomenon: inhibition of membrane function.

Autoradiography of Trypanosoma lewisi labeled in vivo with ³H-thymidine (³HTdR) shows that the shortest doubling time for labeled organisms is 8 hr in intact and immunosuppressed rats. The parasite doubling time increases progressively after the fourth day of infection to 12 hr in immunosuppressed rats and to 24 hr or more in intact rats. The number of days following infection during which the trypanosomes reproduce is prolonged in immunosuppressed rats. In vitro studies of ablastin using ³HTdR-labeled trypanosomes confirmed that cell reproduction halts in the presence of ablastin, but resumes when the parasites are removed from the antibody. Several lines of evidence have been obtained, indicating that the primary effects of ablastin may be on membrane function. Thus, the saturable component for glucose transport in reproducing and ablastin inhibited trypanosomes has an average K_m value of 2.8 × 10^?4M, but the average V_max values for glucose transport are reduced from 3.15 nmole/min/1.25 × 10⁷ reproducing parasites to an average of 1.8 nmole/min/1.25 × 10⁷ nonreproducing forms. Glucose transport is competitively inhibited by 2-deoxyd-glucose (2DOG). The exit and counterflow of ¹⁶C-2DOG from previously loaded trypanosomes is restricted in the presence of antiserum.     

The limitation of growth of Trypanosoma musculi in vitro

Trypanosoma musculi grow readily in vitro provided their growth is supported by mammalian cells. In the presence of murine spleen cells, or spleen cell-conditioned medium, the parasites increase by 100-fold, or more, in a period of 5–6 days. Growth ceases abruptly and death of the parasites soon follows. The reason for the termination of growth has been obscure and is the subject of this report. Termination of growth is not due to an immunological process; not even of ablastin affecting epimastigote reproduction. Instead it appears that other growth inhibitory substances are responsible. Culture medium, collected from spent cultures on day 8 after initiation, inhibits T. musculi growth in fresh medium in dose-dependent fashion. No inhibitory substances were present in medium collected earlier, during the phase of rapid parasite growth. These inhibitory substances appeared to be derived from the parasites rather than the cocultivated spleen cells.     

Trypanosoma lewisi Infections in Normal Rats and in Rats Treated with Dexamethasone

SYNOPSIS. Administration of dexamethasone to rats infected with Trypanosoma lewisi resulted in the development of exceedingly large populations of trypanosomes which were fatal to their hosts. The elevated levels of parasitemia in treated rats early in infections were thought not to be a result of an increased reproductive rate. However, trypanosomes in treated rats 2 days postinfection did have a higher coefficient of variation in total length and a greater percentage of dividing forms than those observed from infected rats which were not given the drug. The course of infection may be markedly altered not only in intensity but also in length by this corticosteroid. It is suggested that dexamethasone administered at the levels recorded to rats infected with T. lewisi inhibits the production of ablastin and trypanocidal antibodies.     

The specific anti-parasite immune responses of germ-free and conventional rats infected with Trypanosoma lewisi

To test the hypothesis that the rapid immune response of rats to Trypanosoma lewisi is elicited by prior exposure to cross-reacting environmental antigens, the early immune response to infection with this nonpathogenic protozoan was studied in germ-free and conventional rats. In germ-free rats, initial levels of both IgG and IgM were significantly lower than those of conventional rats. After infection, the germ-free rats made more immunoglobulins of both classes, and made them more quickly, than did conventional rats. Trypanosome-specific antibodies appeared earlier and in higher titers in the germ-free rats. Because they lacked intestinal microflora, it is unlikely that the germ-free rats' responses had been primed; thus, these observations indicated that the conventional rats' responses to some trypanosome antigens had been down-regulated by their prior exposure to environmental antigens. However, protective antibodies that inhibited parasite reproduction (ablastin) may have been primed, because these appeared in sera 2 days earlier in conventional rats. Despite much lower rates of production of trypanosome-specific antibodies, the conventional rats had the same peak parasitemias and times to crisis as germ-free rats. Thus it is apparent that protective immunity to this nonpathogenic parasite is not down-regulated by prior exposure to environmental antigens, as would be predicted.     

Proteins of the succus entericus from the jejunum of the sheep

1. Suitable methods for studying the proteins passing into the small intestine are discussed. 2. The proteins passing into temporarily isolated jejunal loops between double re-entrant fistulae in four sheep were studied. 3. Loops about 60-70cm. long secreted protein at a rate of 1-5g./24hr. The effect of slight stimulation of secretion by air pressure on the output of protein in 24hr. was not regular. The total protein in the fluid part of the succus entericus is about 2(1/2) times the serum albumin content of the fluid. 4. The additional protein contained in the cellular debris amounts to about 60% of the protein in the fluid part of the succus entericus. 5. Comparison of the proteins in succus entericus with those in serum by immunoelectrophoretic and other electrophoretic methods showed eight components in the fluid part of the succus entericus that appeared to be the same as those in serum and two components that appeared not to be present in serum. 6. Thin-layer gel chromatography in Sephadex G-200 and sedimentation analysis showed that the succus entericus contained two proteins not present in serum, one with sedimentation coefficient (uncorrected) 10s and one sedimenting slower than albumin: they move with the macroglobulin and slower than albumin respectively on gel chromatography. 7. These proteins could be secreted by the glandular epithelium of the small intestine or liberated from desquamated epithelial cells.     

Properties of the transferrin associated with rat intestinal mucosa

The transferrin that is isolated from washed intestinal mucosal cell preparations consists partly of a fraction that has properties distinguishing it from serum transferrin. The serum transferrin contaminating mucosal preparations, even when fully saturated with iron and in the presence of proteinase inhibitors, also acquires the properties of the mucosal transferrin when the mucosa is homogenised. The mucosal transferrin is modified by a single cleavage of the polypeptide chain yielding a disulphide-linked peptide of 6550 daltons linked to the parent protein by a disulphide bridge. The amino-terminal sequence of the first 11 residues of this peptide could be aligned with both the known rat and human transferrin carboxy-terminal sequences. In both cases the sequence is preceded by a phenylalanine residue (residue 622 of human transferrin). This suggested that a mucosal chymotryptic enzyme was responsible even though rat transferrin is not susceptible to alpha-chymotrypsin if fully iron-saturated. Since transferrin mRNA is not found in the intestinal mucosa it must be imported from the serum. It remains uncertain whether the modified transferrin is present naturally and plays a role in iron absorption but these findings do indicate the eventual fate of any transferrin imported into an intestinal cell.     

Insulin-like growth factor binding proteins of equine serum.

Ligand blotting analysis of serum from the horse using radiolabelled IGF-I revealed a protein at 96 kDa which was not present in serum from goat, cow, sheep, deer or donkey. These latter species all displayed five labelled bands in the range 24 to 41 kDa. Conversely, these were only weakly labelled in serum from the horse. Size exclusion chromatography of horse serum pre-incubated with radiolabelled IGF-I revealed reduced binding in the 130-kDa peak compared with goat plasma, and ligand blotting analysis indicated the 96-kDa protein was present in this peak. The 96-kDa protein from horse serum binds IGF-I and IGF-II specifically and appears to be unique to this species. The nature of this protein is at present unknown.     

Sources of the proteins of rat bile

The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component.The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an α₂-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.     

Purification from human pregnancy serum of a low molecular weight mitogen similar to placental lactogen and growth hormone

Recently, we isolated from the serum of pregnant women a factor that induced rapid proliferation of a lactogen-dependent rat lymphoma cell line (Nb2). This mitogenic factor is reasonably specific to pregnancy, since it was present in serum samples from second trimester as well as term-pregnant women, but not in those of adult men or cycling females. It is unlikely that this mitogenic activity (referred to as pregnancy mitogen [PM]) is due to contamination by classical lactogens, since acetone fractionation of serum yielded a preparation devoid of placental lactogen and prolactin, as determined by radioimmunoassays. Further purification of acetone precipitates from term-pregnant serum by ion exchange chromatography and gel filtration yielded a mitogenic activity with a relative mol wt of approximately 10,000. PM activity in the NB2 cell bioassay was not affected by the presence of prolactin antiserum. However, its activity was immunoneutralized by coincubation with anti-placental lactogen serum and, to a lesser extent, anti-growth hormone serum. It appears that PM was not generated by our extraction procedure, since gel filtration of whole serum also yielded a bioactive fraction of approximately 10 kDa. PM was further purified to homogeneity by high-performance liquid chromatography. Examination of the preliminary amino acid composition of PM revealed differences from that of a bioactive fragment of growth hormone and a corresponding portion of placental lactogen, suggesting that PM could be either a molecular variant of these hormones or a novel protein.     

Determination of the rheumatoid factor in gouty patients

The authors have examined the incidence of the rheumatoid factor in the serum of a group of 100 patients, 95 males and 5 females, suffering from gout, of whom only 6 suffered from chronic gout. The rheumatoid factor in the serum was measured by RA-test (1:40) and by Waaler-Rose test (1:32). The rheumatoid factor was not present in the examined subjects and so we can state that such serum parameter does not represent, for its negativity, a laboratory index of any evidence in the gout and it doesn't represent a reason for diagnostic doubts with other forms with the rheumatoid factor in the serum.     

Flow cytometric analysis of immunoglobulin and complement component C3 on the surface of Trypanosoma lewisi

We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab')2 fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body gamma-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasite. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.     

Recombinant antibodies for the depletion of abundant proteins from human serum

The identification of biomarkers from serum or plasma is often hindered by a few proteins present at high concentrations, which may obscure less abundant proteins. Ideal serum depletion strategies would be flexible as regards the proteins to be removed, and would feature the use of reagents with long shelf-lives. In this article, we describe a novel protein depletion methodology based on the incubation of serum samples with phage-derived recombinant antibody fragments, which are able to bind to staphylococcal Protein A, and which carry a C-terminal peptide tag capable of streptavidin binding. The resulting protein-antibody complexes can be removed by simultaneous capture on Protein A and/or streptavidin resin. The depletion methodology was exemplified by the isolation of recombinant human mAb fragments specific to abundant human serum Ags and by the simultaneous depletion of albumin, immunoglobulins, alpha2-macroglobulin, hemoglobin, transferrin and haptoglobin. The depleted serum samples were analyzed by 2-DE and by gel-free MS-based methodologies, confirming the efficiency and selectivity of the depletion process. The methodology presented is modular in nature, since several recombinant antibodies can be combined in a single depletion experiment. Furthermore, antibodies do not have to be covalently coupled to a solid support facilitating long-term storage.     

Biochemical characterization of the serum fetuin-mineral complex

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9-24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.     

Cytotoxic T cells in the peritoneal cavity of mice infected with ectromelia virus.

Peritoneal exudate cells from mice infected with ectromelia virus were cytotoxic for virus-infected target cells as measured in a 51Cr release assay. Cytotoxic activity seemed to be T cell-dependent as it was largely abolished by treatment with anti-theta serum and complement but was not impaired by macrophage depletion. The kinetics of development of cytotoxicity in the peritoneal cavity lagged behind spleen cytotoxicity by 1-2 days. Peak activity in peritoneal cells was present about 6 days after intravenous infection with virus. These studies suggest that macrophages present in the free peritoneal cell populations of ectromelia-infected mice are not cytotoxic for virus-infected target cells. The effect of macrophages in virus clearance is therefore likely to be due to phagocytic rather than cytotoxic effects.     

The contribution of serum triacylglycerol to hepatic triacylglycerol turnover in the starved rat.

The present study was undertaken to evaluate quantitatively the turnover of serum triacylglycerol (triglyceride) in the starved rat and to determine whether serum triacylglycerol recycled to liver contributes a significant fraction of the total hepatic triacylglycerol turnover. Serum was labelled in vitro with [3H]trioleoylglycerol (glycerol [3H]trioleate) to provide uniform labelling of all lipoprotein species. By using the curves describing disappearance of isotope from serum and its appearance in liver, rate constants for movement of triacylglycerol out of serum (0.29 min-1) and the uptake of serum triacylglycerol by liver (0.22 min-1) were calculated. The total rate of movement (flux) of triacylglycerol in these processes, the product of rate constant and serum pool size, was calculated to be 0.39 and 0.29 mg/min per 100 g body wt. respectively. A model is postulated for whole-body triacylglycerol metabolism consistent with the present data as well as most observations in the literature. From the model it can be predicted that: (1) the entire turnover of liver triacylglycerol in the starved rat can be accounted for on the basis of contributions from serum non-esterified fatty acid and serum triacylglycerol; (2) the entire turnover of the serum triacylglycerol pool can be accounted for quantitatively on the basis of contributions from intestine and liver; (3) the release rate for triacylglycerol from liver should be 0.34 to 0.35 mg/min per 100 g body wt.; (4) triacylglycerol synthesized by liver from non-esterified fatty acid of serum and by intestine can account quantitatively for the irreversible disposal rate of triacylglycerol from serum.