PubMed Health⌕ Search

Biomedical subjects

P Diffley

Publications and source records attributed to P Diffley.

18 recordsLinked to original sources
PubMed1997

Kinetic analysis and multiple component monitoring of effectors of adenylyl cyclase activity by quantitative fast-atom bombardment mass spectrometry.

The enzyme adenylyl cyclase catalyses the conversion of adenosine 5'-triphosphate (ATP) to adenosine-3',5'-cyclic monophosphate (cyclic AMP), and is an important pharmaceutical target. Quantitation of this enzyme's activity has been carried out by positive-ion fast-atom bombardment mass spectrometric analysis of the enzyme incubation mixture after the reaction has been terminated. The kinetic data obtained are in good agreement with those obtained by the conventional radiometric assay, and this mass spectrometry-based assay offers the facility to monitor the turnover of several components of the incubation simultaneously. This is utilized to study the relative efficiencies of two ATP-regenerating systems, three phosphodiesterase inhibitors and two modified substrates, and to monitor the uptake and conversion of two competing substrates, adenosine 5' triphosphate and 2'-deoxyadenosine-5-triphosphate, to cyclic AMP and to cyclic deoxyAMP, respectively.

Adenosine Triphosphate↗
PubMed1990

The kinetics of gene expression and maturation of IL-1 alpha after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide.

The purpose of this study was threefold: to determine if the variant surface coat glycoprotein (VSG) of Trypanosoma brucei rhodesiense induces IL-1 alpha; to study the kinetics of IL-1 alpha transcription, maturation and secretion; and to compare VSG to LPS in its ability to induce IL-1 alpha. VSG was added to cultures of the P388D1 murine macrophage cell line. RNA was dotted onto nitrocellulose and hybridized with a murine IL-1 alpha cDNA probe. Maximal production of IL-1 alpha mRNA occurred in a dose- and time-dependent manner, peaking at 25 micrograms/ml VSG, within 2 h. Induction of IL-1 alpha was not due to contaminants because 1) absorption of VSG with a mAb abrogated IL-1 alpha mRNA synthesis, 2) the addition of polymyxin B did not affect mRNA levels, and 3) cellular IL-1 alpha was detectable in VSG-treated splenocytes from endotoxin nonresponder C3H/HeJ mice. Murine splenic macrophages also had enhanced levels of IL-1 alpha mRNA after administration of VSG in vivo or during an acute infection. Antiserum generated against the synthetic peptide SGDDSKYPV (amino acids 177-185 from the murine IL-1 alpha sequence) was used to measure the levels of the 33-, 22-, and 14-kDa proteins in cell lysates and medium of VSG-stimulated P388D1 cells. The 22-kDa protein was the predominant cellular form until secretion started. Secretion of the 14-kDa form began abruptly 6 to 8 h after the addition of VSG. By 12 h, the 33-kDa precursor was the major cytoplasmic form. In comparative analyses, LPS-stimulated P388D1 cells produced more transcript, generated peak levels of 22-kDa protein 3 h earlier, and began to secrete the 14-kDa molecule 5 h earlier. The rate of IL-1 alpha accumulation in the medium was linear between 6 and 24 h after LPS treatment, but began to drop by 8 h in VSG-treated cells. Functional (comitogenic) IL-1 activity was also detected in media from VSG-treated splenic macrophages and P388D1 cells. Activity peaked at 50 micrograms/ml and was lost if 0.2% IL-1 antisera were added to the cultures.

Animals↗
PubMed1989

Fixed and temporary fluctuations in the cell cycle of monomorphic lines of Trypanosoma brucei gambiense.

The growth rate of a cloned, monomorphic strain of Trypanosoma brucei gambiense can be changed in both fixed and environmentally induced ways. The purpose of this study was to determine which phases of the cell cycle were affected by these changes in generation time. A slow-growing cloned line of T. b. gambiense and its fast growing, cloned derivative, were collected from immunosuppressed mice at low parasitemias. The percentages of cells in G1, S, and G2-M phases were determined by cytophotometric measurements of nuclear DNA. The percentages of cells in late G2-M and D phases were established by light microscopy. Over 70% of the fixed reduction of generation time in the cell cycle of the fast growing trypanosome occurred during G1, the remainder in G2. Apparently, the parasite had genetically altered a metabolic pathway necessary for both the initiation of DNA replication and karyokinesis. Both fast and slow growing trypanosomes significantly decrease their growth rates at high parasitemia levels in immunosuppressed rodents. To determine which phases of the cell cycle were affected, nuclear DNA of T. b. gambiense collected at low and high parasitemias were compared. Over 60% of the increase occurred during the S-phase, the remainder in G1. Apparently, DNA replication was affected by the parasite load. Finally, these monomorphic strains of T. b. gambiense, like tumors, apparently do not have a noncycling (G0) phase or a restriction point in G1 since slow growing trypanosomes in either optimal or suboptimal conditions did not accumulate in a diploid state or proceed through S to D phases in a constant length of time.

Animals↗
PubMed1987

The rate of proliferation among African trypanosomes is a stable trait that is directly related to virulence.

The relationship between the growth rate of Trypanosoma brucei gambiense and its virulence was investigated. A cloned, monomorphic, slow growing, and relatively avirulent line of T. b. gambiense was serially passaged at 3- to 5-day intervals through immunosuppressed mice. The growth rate measured within the first 2 patent days of infection did not vary significantly through the first 25 passages but by passage 50 had decreased significantly from 11.9 +/- 1.1 hr to 9.0 +/- 0.7 hr. A clone from passage 50 and three different second peak heterologous variants all had statistically similar growth rates, indicating that the rate of proliferation was a stable trait. With the faster rate of proliferation there was a corresponding increase in virulence. The inoculum necessary to kill 50% of normal outbred mice in the first peak of parasitemia (LD50) dropped significantly from 3 X 10(6) first passage parasites to 4 X 10(5) passage 50 parasites. The lethal load for both fast and slow growing organisms was the same (greater than 2 X 10(9) trypanosomes/ml of blood). To further link virulence and growth rate, a strong correlation (r = 0.89) was measured when generation times of 10 closely related lines of T. b. gambiense, and 2 lines of pleomorphic T. b. rhodesiense were compared to their LD50 values. While the rate of trypanosomal proliferation was similar between the day of inoculation through the second patent day, it slowed to 64% of that level once parasitemias exceeded 3 X 10(8) organisms/ml of host blood.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Influence of source and quantity of protein on the development of immunity and resistance to African trypanosomiasis.

Although it is well documented that severe protein deprivation inhibits the development of the immune response and exacerbates certain infections, little has been done to study the effects of native diets on endemic diseases or immunity. Therefore, protein-restricted diets were formulated for mice to mimic the sources and amounts measured in human diets of the Batouri region of Cameroon, endemic for African trypanosomiasis. Weanling C57BL/6 female mice were fed a diet that contained 73% of the recommended daily allowance (RDA) of protein. The sources of protein were all plant (cornmeal), all animal (casein), or a ratio that reflected the native diet (2.2 parts plant to 1 part animal protein). Diets were isocaloric on a weight basis, equal in lipids, and adequate in vitamins and minerals. Control mice were fed laboratory chow or two times the RDA of animal protein (casein). Mice fed only cornmeal or the native diets consumed as much food but did not gain as much weight as mice fed only animal protein, indicating the poorer quality of protein in their diets. Upon infection with Trypanosoma brucei gambiense, however, significantly higher numbers of these mice controlled the first peak of parasitemia and survived the infection as compared with mice fed the other three diets. Since all mice developed patent infections and the parasite growth rate was unaffected by diet, innate immune factors were ruled out as the cause for the higher level of resistance to the parasite. To determine whether diet affected the development of the immune system, weanling mice were maintained on diets for 30 days before immunization with sheep erythrocytes or trinitrophenylated Ficoll. Mice fed only plant protein or native diets elicited higher direct plaque-forming-cell responses to both the T-cell-dependent and T-cell-independent antigens. Since variant-specific immunity which controls levels of African trypanosomes in the blood is a T-cell-independent humoral immunoglobulin M response, this suggests that cornmeal, a protein of poor quality, was adequate for the development of humoral immunity and resistance to African trypanosomiasis while casein, an animal protein of high quality, was not. This provides more evidence that diet plays an important role in infection and immunity.

Animals↗
PubMed1986

Biochemical and immunological characterization of the variant surface coat glycoprotein shed by African trypanosomes.

As the variant surface coat glycoprotein (VSG) was shed from Trypanosoma brucei rhodesiense into the blood of infected rats, it was biochemically characterized and compared with VSG that had been purified from trypanosomal homogenates. To determine if VSG was in association with lipid, membranes and lipoproteins in plasma of infected rats (IRP), VSG isolated from plasma (PVSG), and VSG isolated from trypanosomal homogenates (HVSG) were all concentrated by ultracentrifugation and assayed for the presence of VSG by radial immunodiffusion (minimum level of detection, 25 micrograms/ml) and by immunoelectroblots (minimum level of detection, 1 microgram/ml). Crimson red was used to detect lipid (minimum level of detection, 10 micrograms per sample) in electrophoresed samples. The VSG was neither concentrated with membrane or lipoprotein fractions nor stained by lipid crimson. Lipids from normal rat plasma, IRP, trypanosomal homogenates, HVSG, and PVSG were also extracted and separated by thin-layer chromatography (minimum level of detection, 20 micrograms of trypanosomal phospholipid per sample). The trypanosomal homogenates had five bands as detected by iodine vapors, of which three were phospholipids as detected by molybdenum blue. Both normal rat plasma and IRP had identical patterns of bands with a single phospholipid. The PVSG had one neutral lipid contaminant that apparently was not physically associated with the shed surface coat. The HVSG contained no lipids at all. Therefore, no evidence was obtained to implicate an association between membranes and VSG, once the latter had been shed into the blood of infected hosts. From immunoelectroblots of denatured material, it was determined that both HVSG and PVSG had the same reduced molecular weight. From molecular sieve column chromatography, however, it was determined that VSG released during the homogenization of trypanosomes is a noncovalently linked dimer, whereas that shed in the blood is apparently a trimer. This difference in native structure made no difference in immunological effect. Administered in a regimen that mimicked what the host encounters during a first peak of parasitemia, both HVSG and PVSG induced nonspecific proliferation of splenic lymphocytes and production of unelicited antibodies without the generation of nonspecific immunosuppression. This polyclonal activation of lymphocytes was not the result of contamination by exogenous pyrogen, because the activity was lost if VSG was immunologically absorbed from plasma.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Mice varying in resistance to African trypanosomiasis respond differently to treatments with variant surface glycoprotein.

A comparative analysis of responses between resistant and susceptible hosts revealed that DBA/2 mice, after treatment with variant surface coat glycoprotein (VSG) from virulent or avirulent African trypanosomes, developed splenomegaly as the result of a near-doubling of the splenic cell population, had less polyclonal activation of B cells and were protected upon challenge with homologous trypanosomes. The susceptible C3H/Anf and C3H/HeJ mice on the other hand increased their splenic cell population by only 12%, had about twice the production of unelicited antibodies and were not immunized by the VSG treatments. This indicated that (a) proliferation of spleen cells during African trypanosomiasis may reflect an attempt to generate a specific and protective immune response and is not merely the result of polyclonal activation of lymphocytes; (b) production of unelicited antibodies is not merely a "bystander reaction" to the generation of antigen-specific responses; and (c) polyclonal antibody production in response to VSG is not linked to the LPS gene. Nonspecific immunosuppression as measured in mitogen assays was not elicited by VSG in either resistant or susceptible mice, indicating that polyclonal lymphocyte activation and nonspecific immunosuppression are unlinked phenomena. Mice injected with VSG from either virulent or avirulent isolates at levels normally encountered by hosts during severe, acute infection developed the same degree of splenomegaly and production of unelicited (polyclonal) antibodies. Therefore, any differences in polyclonal activation of lymphocytes measured between mice with acute vs. chronic African trypanosomiasis can be attributed to quantitative and not qualitative differences in VSG.

Animals↗
PubMed1985

Trypanosoma brucei: immunogenicity of the variant surface coat glycoprotein of virulent and avirulent subspecies.

Comparative analyses were made to define the immunogenic role in mice of the variant surface coat glycoprotein (VSG) of African trypanosomes. Less than 10 micrograms of the glycoprotein fixed to trypanosomes or covalently linked to sheep erythrocytes were 100 times more immunogenic than soluble VSG. Therefore, although VSG is present on the parasites and in the blood of infected hosts, the cell-bound form most likely elicits immunity. Intravenous administration of soluble or cell-bound VSG was a better route of immunization than the subcutaneous route. Therefore, although parasites grow at the site of infection, in tissue spaces, and in the blood, control of blood parasitemia is best developed if the antigen is introduced to the vascular bed. Full protection against homologous challenge occurred by 4 days and was maintained through 30 days. Trypanosome-agglutinating antibody titers could be measured at 3 days, peaked at 5 days, and remained high through 14 days after immunization. Therefore, mice immunized with an optimal dosage of VSG, 2 days before challenge, should have had ample time to elicit a protective response. Most of these mice, however, developed patent infections, and one-third died during the first peak of parasitemia at about the same time as untreated control mice. This indicates that active infection inhibits the early phases of induction of immunity. Mice, suboptimally immunized against and challenged with an avirulent isolate of Trypanosoma brucei gambiense, survived at higher rates than mice immunized and challenged with a virulent clone of T. b. rhodesiense. Cell-fixed and soluble VSG from both parasites elicited similar agglutinating-antibody titers, indicating that the two trypanosomes were equally antigenic. Results from neutralization tests, however, revealed that, per unit of immune mouse serum, 400 times more T. b. gambiense became noninfective than T. b. rhodesiense. Apparently, virulence is related to relative sensitivity of the trypanosomes to immunological assault.

Agglutination Tests↗
PubMed1984

Immunological control of chronic Trypanosoma brucei gambiense in outbred rodents.

Recent human isolates of Trypanosoma brucei gambiense generally fail to become or remain patent in laboratory rodents. The purpose of this study was to determine if this was due to acquired immunity and if so which immunosuppressive method was the most efficient in raising parasitemia levels. Prior to infection, rats and mice were immunosuppressed by treatments with cobra venom factor, anti-lymphocyte sera, hydrocortisone acetate, cyclophosphamide; by splenectomy; or by lethal X-irradiation. While no parasites were detected in the blood of most of the untreated rodents for 30 days postinfection, all immunosuppressive procedures resulted in patent parasitemias in at least fifty percent of the treated animals. The most effective method, lethal X-irradiation, consistently caused fulminating infections typical of acute African trypanosomiasis. Cyclophosphamide had the same effect as X-irradiation in rats but was less effective in mice. Splenectomy allowed fulminating first peak parasitemias in two-thirds of the rodents while cobra venom factor and anti-lymphocyte sera in general allowed only low first peaks of parasitemia that were resolved within 10 days of infection. Hydrocortisone acetate allowed low grade and sporadically patent infections throughout the 30-day study. To determine if in untreated rodents, the parasites were eliminated or maintained in a subpatent state, rodents infected for 30-45 days were immunosuppressed with cyclophosphamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Trypanosomal surface coat variant antigen causes polyclonal lymphocyte activation.

Variant antigen, the primary component of the surface coat of the salivarian trypanosome, when injected i.v. into mice at dosages encountered during acute infections, produced some of the immunopathogenic phenomena associated with acute African trypanosomiasis. Trypanosome-infected and variant antigen-treated mice had splenomegaly caused by proliferation of null cells, B and T lymphocytes, and macrophages. Splenic B lymphocytes were nonspecifically activated to produce antibodies to sheep erythrocytes (SE), to trinitrophenylated-SE, and to fluoresceinated-SE. This polyclonal activation apparently depleted antigen-reactive lymphocyte populations as indicated by a reduced SE-specific, plaque-forming cell response after immunization in vitro. As judged from the results of in vitro immunization and mitogen assays, injections of variant antigen did not induce nonspecific immunosuppression, which indicated that the polyclonal B cell activation and nonspecific immunosuppression may be uncoupled events induced by different parasite products. The polyclonal lymphocyte activation was induced with variant antigen isolated by either ion exchange or lentil lectin column chromatography and by variant antigen isolated from antigenically dissimilar Trypanosoma brucei rhodesiense and Trypanosoma brucei brucei clones.

Absorption↗
PubMed1982

Surface coat variant antigen of Trypanosoma brucei brucei: its clearance from blood and concentration in organs of normal, infected, and immune mice.

Experiments were conducted to determine the fate of variant antigen once it was shed from the surface of Trypanosoma brucei brucei. Radioiodinated variant antigen was administered intravenously to normal mice, mice immunized to the homologous variant antigen, and mice infected with an antigenically dissimilar (heterologous) T. brucei brucei variant. The variant antigen was cleared slightly faster in infected and immune animals. Though over 80% of the variant antigen was cleared in all animals within 4 h, traces of radioactivity could be detected in the peripheral blood at 48 h postinjection. At 1 h postinjection, most of the variant antigen had collected in the liver, kidneys, bone marrow, spleen, lungs, thymus, and lymph nodes (listed in the order of decreasing radioactivity). At 24 h, the kidneys, liver, and spleen retained the most radioactivity. The kidneys had 10 to 20 times, the liver had 8 to 10 times, and the spleen had 5 to 7 times more variant antigen than was present in the organs' blood supply. At 48 h postinjection, two-thirds of the radioactivity present at 24 h remained in these tissues. The livers and spleens from infected animals had, on a per gram of tissue weight basis, reduced uptake of the soluble antigen whereas their lungs had an increased uptake. Radioactivity in blood and organs was proved to be associated with protein by electrophoresis/autoradiography and precipitation of radioactive protein of tissue extracts.

Animals↗
PubMed1982

Comparative analysis of procedures used to isolate variant antigen from Trypanosoma brucei rhodesiense.

Comparisons made among various procedures leading to the isolation of variant antigen from Trypanosoma brucei rhodesiense bloodstream trypomastigotes. As a means of parasite disruption, freeze-thawing solubilized 36% more variant antigen than did sonication. Protease inhibitors were important additions to the suspension prior to cellular disruption. If trypanosomal extracts were frozen for at least 1 wk prior to chromatographic isolation of variant antigen, recovery of the antigen was reduced by 70%. Ion exchange chromatography was more efficient in the isolation of variant antigen than either lentil-lectin or antibody-affinity columns. All three methods yielded qualitatively similar variant antigen preparations. Using the most efficient isolation procedure tested, about 4 mg of variant antigen was isolated per 10(10) bloodstream trypomastigotes. The most efficient means of isolating variant antigen from plasma of infected rats began with passage of fresh plasma with protease inhibitors through an ion exchange column followed by antibody-affinity chromatography. This resulted in a preparation that was 52% variant antigen, a 370-fold concentration over plasma levels.

Animals↗
PubMed1980

Detection and quantification of variant specific antigen in the plasma of rats and mice infected with Trypanosoma brucei brucei.

Variant specific antigen (VSA), the principal constituent of the surface coat of salivarian trypanosomes, was detected by gel immunoassays in the plasma of rats and mice infected with Trypanosoma brucei brucei. The quantity of VSA in plasma was measured in radial immunodiffusion tests using a monospecific antiserum and purified VSA as a standard. During the first peak of parasitemia, a statistically significant, linear relationship was determined between the number of parasites in the blood (in the range between 4 x 10(8) and 10(9)/ml) and the concentration of VSA in the plasma (28-320 microgram/ml). The VSA from parasites of the first peak was lost within 2 days of remission. Variant antigens of parasites constituting the second peak then began to appear in the plasma of infected rats. All plasma samples had been separated from parasites and blood cells within 15 min of blood collection. The pH of plasma was controlled with a buffered anticoagulant. No soluble parasite antigens, other than VSA, were detected in the plasma of infected hosts. The results of this study extend the observation that salivarian trypanosomes shed surface coat material during the course of infection. Thus, sloughed VSA may be the parasite product that has been hypothesized to cause the nonspecific lymphocyte proliferation, immunosuppression, and/or hypergammaglobulinemia which occur during African trypanosomiasis.

Animals↗
PubMed1978

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.

Animals↗
PubMed1976

Delayed type hypersensitivity in guinea pig infected subcutaneously with Naegleria fowleri Carter.

A soluble fraction, derived from Naegleria fowleri trophozoites disrupted by freeze-thawing, was tested for antigenic properties. Intradermal injections of this preparation were administered to guinea pigs previously infected subcutaneously with viable N. fowleri. Delayed hypersensitivity to the antigen and loss of weight, the diagnostic symptom of visceral naegleriasis, were observed in the surviving animals. Fifty percent of the guinea pigs, however, did not lose weight and had a reduced reaction to the antigen. The apparent differences in the immunocompetence of guinea pigs inoculated subcutaneously and intranasally with N. fowleri are compared.

Amebiasis↗