13C NMR studies of glycolysis in intra- and extra-erythrocytic Babesia microti

Metabolism in the erythrocytes of normal mice and mice infected with Babesia microti has been monitored non-invasively by high resolution ¹³C NMR spectroscopy. The conversion of [U-¹³C]glucose to lactate in both normal and infected cells together with the effect of the trypanocidal drug, 4,4′-diamidinodiazoaminobenzene diaceturate, on glycolytic rates were monitored. These studies show that erythrocytes utilize [U-¹³C]glucose at a rate of 3 × 10^?12μmol cell^?1 min^?1 at 35°C while parasitized cells consume 2.9 × 10^?11μmol cell^?1 min^?1 and produce lactate as the sole end-product. This rate decreases to 9 × 10^?12μmol cell^?1 min^?1 on the addition of 0.75 mM drug.     

Radioiodination of new protein antigens on the surface of Plasmodium knowlesi schizont-infected erythrocytes

Schizont-infected red blood cells (SI-RBCs) from Plasmodium knowlesi-infected rhesus monkeys (Macaca mulatta) have been radioiodinated and the ¹²⁵I-proteins and ¹²⁵I-antigens compared with those of uninfected RBCs. New ¹²⁵I-proteins of M_r 230 000, 180 000, 165 000, 155 000, 135 000, 107 000, 72 000 and 65 000 were shown to be parasite-dependent components of SI-RBCs, the M_r 230 000 and 72 000 bands being quantitatively the major new components. New ¹²⁵I-antigens of SI-RBCs that were absent from uninfected cells and were only immunoprecipitated by sera from infected monkeys had M_r 230 000, 200 000, 180 000, 165 000, 155 000, 135 000, 130 000, 107 000, 72 000, 65 000 and 47 000. The following approaches were used to test which new radioiodinated components are on the SI-RBC outer membrane: analysis of radioactivity in haemoglobin; electron microscopic analysis of the integrity and purity of the SI-RBCs; treatment of intact ¹²⁵I-SI-RBCs with trypsin to ascertain the sensitivity of new proteins to exogenous protease; immunoprecipitation of antigen-antibody complexes after addition of antibody to intact ¹²⁵I-SI-RBCs. Although each test has inherent disadvantages, the accumulated evidence suggests that many, if not all of the new antigens identified for the first time in this report are on the SI-RBC outer membrane. The SICA variant antigen on P. knowlesi SI-RBCs was not identified by this approach.     

N-terminal amino acid sequence of the histidine-rich protein from Plasmodium lophurae

We have determined the N-terminal amino acid sequence of the first 25 amino acids of the histidine-rich protein (HisRP) isolated from granules of the avian malaria parasite Plasmodium lophurae. The protein was purified from cytoplasmic granules and shown to be 65.2 mol % histidine, close to the previously described value of 73 mol % histidine (Kilejian (1974) J. Biol. Chem. 249, 4650–4655). Ten of the first 25 residues were histidine, five of which formed the sequence His-His-His-His-His (positions 14–18). Also notable was the presence of eight acidic residues within the N-terminal 25 residues. HisRP contained no detectable carbohydrate. When the HisRP was biosynthetically labeled in cultured infected erythrocytes, incorporation of [³H]His greatly exceeded [³H]Ile. Labeled HisRP was not solubilized with 1% w/v Triton X-100 but could be solubilized with ? 1% w/v sodium dodecyl sulfate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the [³H]His labeled protein migrated as a doublet (M_r 53 000 and 50 000). Only one of these bands (M_r 53 000) comigrated with the Coomassie Blue stained protein isolated by the acid-extraction procedure from purified granules. The amino acid composition of HisRP and presence of five contiguous histidine residues in the sequence studied here suggests that other sequences of several contiguous histidine residues must exist in this molecule.     

Delayed Hypersensitivity to Toxoplasma and Unrelated Antigens in Toxoplasma-Infected Mice: Induction and Elicitation of Delayed-Type Hypersensitivity by Antigen-Pulsed Macrophages

Delayed-type hypersensitivity (DTH) to Toxoplasma and unrelated antigens in Toxoplasma-infected BALB/c mice was investigated by the radioisotopic uptake method of Vadas et al. (Int. Arch. Allergy Appl. Immunol. 49: 670-692, 1975). DTH became positive on day 30 of infection and remained positive during chronic infection. The expression of DTH in mice infected with the relatively avirulent C37 strain of the parasite paralleled the Toxoplasma antibody response as detected by the Sabin-Feldman dye test. Mice sensitized with Toxoplasma, keyhole limpet hemocyanin, or sheep erythrocytes during the acute or chronic phase of Toxoplasma infection showed a DTH reaction similar to that of uninfected sensitized controls. No parasite antigens could be detected by immunofluorescence techniques on the surface of Toxoplasma-infected cells. When killed organisms were added to the cell cultures, specks of fluorescence appeared on cells containing intracellular parasites as well as on cells without intracellular organisms. That the antigens may be present in or on macrophages in a form readily recognizable by T cells is suggested by experiments in which we demonstrated that injection of uninfected normal macrophages pulsed with Toxoplasma-soluble antigens into the ears of chronically infected mice elicited a DTH reaction comparable to that observed when 10⁶ Formalin-fixed tachyzoites were used as the test antigen. When macrophages pulsed with Toxoplasma antigen were used in attempts to induce DTH in naive uninfected mice, the intensity of the reaction was similar to that observed in infected mice.     

A simple method for separation of uninfected erythrocytes from those infected withPlasmodium berghei and for isolation of artificially released parasites

Rat erythrocytes infected withPlasmodium berghei were disrupted by gentle passage of Coneanavalin A (ConA) agglutinated cells through a 100 mesh stainless steel grid. The free parasites were separated from cell debris, unbroken infected cells, and from uninfected rat erythrocytes on a Percoll gradient. The free parasites remained morphologically intact, metabolically active, and infective to mice.The parasites were observed by light and electron microscopy. The incorporation of³H-isoleucine and³H-hypoxanthine was compared in intact and infected cells, and the infectivity was measured by the injection of parasites into susceptible mice. It seems that the combination of the two techniques used, produces a high yield of intact free parasites suitable for physiological or immunological studies.     

In vivo expression of signaling proteins in reconstituted NK cells

Natural Killer cells are cells of the innate immune system that are important for the recognition and clearance of virally infected cells or tumors. Examination of the development and signaling of these cells has been severely hampered due to an inability to over-express proteins in these cells. We developed a novel technique to generate NK cells in vivo, all of which express a gene of interest. IL2Rγ_c^−/−/Rag2^−/− mice do not develop NK cells due to the lack of IL15 signaling. We infected bone marrow from IL2Rγ_c^−/−/Rag2^−/− mice with a retroviral construct encoding EGFP and IL2Rγ_c connected by an IRES. NK cells selectively developed through expression of IL2Rγ_c and 100% of these NK cells were found to be EGFP⁺. In order to test the utilization of this method to examine the function of biologically relevant proteins, constitutively active PI3K p110γ and p110δ isoforms were over-expressed in this system. Constitutively active p110γ revealed profound effects on NK cell development and function in vivo while p110δ had little effect.     

The role of gene Nu3 in bacteriophage lambda head morphogenesis

The role of gene Nu3 in λ head morphogenesis has been investigated using a recently isolated amber mutant (am_a8) in this gene. Analysis of lysates of sup^? cells infected with λam_a8 using electron microscopy, revealed the presence of a relatively high proportion of “monsters.” In this respect, such lysates resemble those obtained from λBamCam infected cells. The nature of the gene Nu3 product, and its involvement in several interactions with other morphogenetic proteins, were examined by SDS-polyacrylamide gel electrophoresis, followed by autoradiography of radioactively labeled cell extracts. The product of gene Nu3, pNu3³, is a polypeptide of 19,000 molecular weight. It appears to be bound to precursor head structures very early during assembly, but is subsequently released and cleaved. pNu3 is required for the functional assembly and cleavage of the products of genes B and C. In Nu3 mutants neither pB nor pC are found associated with the nascent head structures, known as petit λ. On the basis of these data and of results published by others a model is proposed to account for the interactions that take place among morphogenetic gene-products during the early events in head morphogenesis.     

Immunoselection and characterization of moloney murine leukemia virus-infected cell lines deficient in surface gag antigen expression

NIH/3T3 cells infected with Moloney murine leukemia virus (M-MuLV) which were deficient in gag surface antigen were selected by incubation with anti-serum to the major gag virion protein, p30, in the presence of complement. Survivors of the selection were cloned and characterized with respect to intracellular production of gag and env gene products, gag surface antigen expression as revealed by indirect immunofluorescence, and virus production. Nineteen clones tested were all positive for env gene products in cytoplasmic extracts. Seventeen of the nineteen were positive for gag gene products, and two were negative. The gag-positive clones all produced Pr65^gag (the precursor to the internal structural proteins) and they also produced gPr80^gag (the precursor to the cell-surface gag antigen). The selected clones were all deficient in the presence of surface gag antigen as measured by immunofluorescent microscopy or flow microfluorimetry, and they all processed Pr65^gag to mature p30 more slowly than the parental cells. In addition, all of the surface gag-deficient clones produced virus at a reduced rate. The nature of the defect in one gag-deficient clone was studied by infection of progeny virus onto uninfected NIH/3T3 cells. The resultant cells showed normal gag surface fluorescence and virus production. This suggests that the defect was cellular rather than viral.     

Further studies on Chara corallina virus

The particles of Chara corallina virus (CCV) and those of tobamoviruses share many properties, but differ in some. CCV particles are tubular, 532 nm long, and 18 nm wide, and are helically constructed with a basic pitch of 2.75 nm; their isoelectric point is pH 3.4–3.7; their sedimentation coefficient (s₂₀, w) is 230 S; they contain 5% RNA with a molecular weight of 3.6 × 10⁶ daltons, and with a base ratio of G 24.5, A 28.0, C 20.0, U 27.5; their coat protein is similar in size to that of tobamoviruses (about 17.5 × 10³ daltons). CCV is considered to be a tobamovirus. CCV may be transmitted experimentally to uninfected Chara corallina cells by injection of virus particles, and causes chlorosis and death in about 10–12 days. A classification of CCV and 68 other tobamovirus isolates, whose coat protein composition is known, showed that CCV is distant from all, but is most closely related to the cucurbit tobamoviruses.     

f1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

The phospholipid composition of cytoplasmic membranes prepared from bacteria grown in the presence of [³²P]phosphate and infected with f1 wild type and f1 amber mutant bacteriophages was determined. Ninety minutes after infection with f1 amber mutants in genes 1, 3, 4, 5, 6, and 7 the percentage of cardiolipin was increased from the level in uninfected bacteria of 5% to about 20–35%, and the percentage of phosphatidylethanolamine was decreased from 70% to about 50–60%. The phospholipid composition of cytoplasmic membranes from bacteria infected with a phage containing an amber mutation in the coat cistron (gene 8) did not differ from that of uninfected bacteria. Although late in infection there were no detectable alterations in phospholipid metabolism in wild type infected bacteria, transient alterations in phospholipid metabolism occurred in these bacteria 10 to 20 min after infection. During this time period, the f1 coat protein was found to be rapidly synthesized but was not being packaged into mature phage and released from bacteria. Both the long-term alterations of phospholipid metabolism found in the amber mutant infected bacteria and the transient alterations found in wild-type infected bacteria resulted from an increase in the rate of synthesis of phosphatidylglycerol and cardiolipin and a decrease in the rate of synthesis of phosphatidylethanolamine. These results are discussed in terms of the relationship between the accumulation of f1 coat protein in infected bacteria and the observed alterations in phospholipid metabolism.     

Comparative study of IgA VH3 gene usage in healthy TST− and TST+ population exposed to tuberculosis: deep sequencing analysis

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST⁺) and uninfected TST⁻) with M. tuberculosis. From a total of 22 V genes analysed, the TST⁻ population preferred the V_H3-23 and Vκ1 genes. The V_H3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST⁻ population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST⁺ population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST⁻ population. The antibodyome difference between both populations suggests a preference for antibodies with V_H3-23, D3-3, J_H4 gene usage by the TST⁻ population that could be associated with resistance to infection with M. tuberculosis.     

Agglutination of Mouse Erythrocytes by Eperythrozoon coccoides

Erythrocytes from blood of mice infected with Eperythrozoon coccoides for 3 or 4 days agglutinated spontaneously. Washed E. coccoides particles agglutinated washed erythrocytes of uninfected mice. E. coccoides-mediated agglutination of normal mouse erythrocytes would be an excellent system for studies of bacterial adhesion.     

FOXP3 expression and frequency of regulatory T cells in healed individuals from Leishmania major infection and the asymptomatic cases

Two groups of residents in an endemic area of Leishmania major infection in Iran with positive leishmanin skin tests who were either asymptomatic or had healed cutaneous leishmaniasis lesions were compared with respect to their T helper responses. The percentages of regulatory T cells (T_reg; CD4⁺CD25^high FoxP3⁺) from the peripheral blood and CD4⁺ T cells producing intracellular cytokines (IL-4, IL-10, IL-17 and IFN-γ) from the stimulated PBMCs were evaluated by flow cytometry and the expressions of RORC and FOXP3 genes were quantified by real-time RT-PCR. T responder (CD4⁺CD25⁻) and T_reg-enriched (CD4⁺CD25⁺) cells were isolated magnetically and the suppressive capacity of the latter and the cytokines (IFN-γ, TGF-β and IL-10) secreted from them were evaluated by in vitro assays. The results showed that the frequency of T_reg in the studied groups were similar and T_reg from both groups exhibited high yet similar suppressive capacities while significantly higher levels of FOXP3 expression was observed in the asymptomatic group. Taken together, similar frequency and suppressiveness of T_reg combined with high ratios of IFN-γ/IL-10 producing CD4⁺ T cells were common in both groups; however the members of the asymptomatic group appeared to require higher expression of FOXP3 to maintain their immunity to re-infection.     

Intestinal epithelial membrane changes in rats immune to Trichinella spiralis

Establishment of Trichinella spiralis infective larvae is blocked to a large degree in the immune rat as compared with the nonimmune host. The rapidity with which this response occurs indicates that most worms are either prevented from penetrating the intestinal epithelium or are rejected immediately after cell entry. It is proposed that interference with larval infectivity is due to alterations in the epithelial cell apical or brush border membrane. Alterations may result from prior infection or may reflect an acute change induced by challenge infection. In either case the establishment of normal populations of larvae in the mucosa is disturbed. Lectin binding capacity of brush border membranes was used to assess possible membrane alterations. This parameter in uninfected (control) rats was compared with that in infected rats, which acquire resistance to subsequent challenge, and in infected rats immediately after a challenge inoculum. Enriched brush border membrane preparations were characterized for their binding of wheat germ agglutinin, which attaches specifically to the carbohydrate, N-acetylglucosamine. Maximum specific binding of ¹²⁵I-labeled wheat germ agglutinin occurred within 20 min. The spontaneous rate of dissociation was negligible for 90 min. Highest specific binding resulted at 24°C, pH 6.0 and with 75 μg bursh border membrane protein per assay tube. Results suggested the existence of multiple binding sites. 1 mg of membrane protein from uninfected rats and rats immunized by primary infection maximally bound 9.8 × 10¹⁰ and 4.3 × 10¹⁰ molecules of wheat germ agglutinin, respectively. Binding for the ‘immune’ brush border membrane, as compared with the ‘uninfected’ brush border membrane was reduced during the first 3 weeks of infection and remained low for at least 3 months. No further reduction in binding was observed for brush border membrane isolated within minutes after a secondary infection. These results reveal the induction by a primary infection of changes in brush border membrane structure and the persistance of these changes in the immune host. In view of the rapid turnover time of epithelial tissue the mechanism by which this change is perpetuated speculatively involves immune elements in the lamina propria.     

Identification of a schizont- and species-specific surface glycoprotein on erythrocytes infected with rodent malarias

Erythrocytes infected with mature trophozoites of Plasmodium chabaudi and reticulocytes infected with P. berghei were labelled metabolically in vitro with [³⁵S]methionine. The labelled cells were incubated with normal and immune serum and washed to remove unbound antibody. Solubilisation of the antibody-coated cells in detergent was followed by co-precipitation of antibody/antigen complexes and analysis of the immunoprecipitates by SDS-PAGE and fluorography. One major parasite polypeptide of 250 000 daltons was found to be exposed to antibody in both species. A labelled band of the same molecular weight could be identified by immunoprecipitation and SDS-PAGE analysis of P. chabaudi-infected cells that had been surface-labelled with periodate/NaB³H₄. This molecule also incorporated [³H]glucosamine in short term cultures of mature parasitised erythrocytes. The results suggest that a 250 000 dalton glycoprotein which is synthesised only by late trophozoites or schizonts is exposed either on the surface of the infected erythrocyte, the surface of the merozoite, or both. Furthermore, the exposed portion of the molecule was not immunologically cross-reactive in the two Plasmodium species, but some cross reaction was detectable in total parasite lysates. The significance of these findings to protective immunity is discussed.     

Phenotypic Characterization of Splenic T Cells from Mice Infected with Plasmodium chabaudi chabaudi

T cells from spleens of mice infected with the erythrocytic stages of Plasmodium chabaudi chabaudi have been analysed with respect to their expression of surface molecules CD3, CD4 and CDS and T-cell receptor (TCR)αβ and γδ. The majority of T cells from infected mice were αβTCR ⁺. However, there was an increase of approximately 8–10-fold in the proportion and total number of γδ T cells. Immunocytochemical analysis of sections of spleens taken from infected C57BL/6 mice during a primary infection showed that this increase took place particularly in the non-lymphoid areas. Within the αβ TCR⁺ T-cell population, both CD4⁺ T cells and CD8⁺ T cells were represented in proportions similar to those observed in normal uninfected mice. Stimulation of splenic T cells from infected mice with P. chabaudi-infected erythrocytes in vitro resulted ina blasted cell population composed predominantly of αβTTCR⁺ T cells with no preferential expansion of γβTCR⁺ T cells. There was no evidence of superantigen-like stimulation of T cells bearing particular Vβ chains of the TCR. The representation of the different Vβ chains within the population was not significantly different from that seen in uninfected mice.     

Dependence of herpes simplex virus type 1-induced cell fusion on cell type

Syncytial mutants of herpes simplex virus type 1 (HSV-1), such as syn20, cause extensive fusion of human embryonic lung (HEL) cells but only a small amount of fusion of human epidermoid carcinoma No. 2 (HEp-2) cells. In order to determine the cellular basis of this difference in fusion, sparse cultures of syn20-infected HEL or HEp-2 cells, previously labeled with [³H]thymidine, were surrounded with uninfected, unlabeled HEL or HEp-2 cells. The fusion of radioactive with nonradioactive cells was determined at different times after infection using radioautography. syn20-infected HEL cells fused extensively with surrounding uninfected HEL or HEp-2 cells, while syn20-infected HEp-2 cells fused poorly with surrounding uninfected HEL or cells. Therefore, the major difference in the fusion capacity of HEL and HEp-2 cells was not due to a difference in cell-surface receptors for a fusion factor in the two cell types. The process of infection of HEp-2 cells did not cause the plasma membranes of the cells to become refractory to fusion, because syn20-infected HEL cells fused equally well with either uninfected or infected HEp-2 cells. The capacity for a mutant virus to express the syncytial phenotype in mixed infection with a wild-type virus is also dependent on cell type. In a mixed infection with equal numbers of MP and its nonsyncytial parent, mP, extensive fusion was observed for infected HEL cells and significantly less fusion was observed for infected African green monkey kidney (CV-1), baby hamster kidney (BHK-21), and HEp-2 cells.     

Equine herpesvirus-induced alterations in nuclear RNA and DNA polymerase activities.

Hamster hepatic cells infected in vivo with equine herpesvirus, type 1, were used for the preparation of isolated nuclei, which were assayed for RNA and DNA polymerases. An endogenous RNA polymerase II activity (sensitive to α-amanitin) was inhibited early and progressively during infection. The radiolabeled RNA products, synthesized in vitro were characterized by analytical centrifugation in sucrose-formamide gradients and by hybridization analysis. The bulk of the RNA synthesized by uninfected and 9-hr-postinfection (p.i.) nuclei sedimented at 10–18 S and 10–14 S, respectively. Purified RNA from uninfected nuclei hybridized exclusively to hamster DNA, whereas RNA from infected nuclei hybridized predominantly to viral DNA (12% of the input radioactivity measured as counts per minutes) and to a lesser degree to hamster DNA (1% of the input counts). Endogenous DNA polymerase activity, assayed in the presence of 150 mM K₂SO₄, was completely inhibited in uninfected nuclei; however, an induced DNA polymerase, active under the same conditions, was detected as early as 2 hours p.i. Overall, a 35-fold increase in activity of the “high salt DNA polymerase” was noted between 2 and 9 hr p.i. Radiolabeled DNA synthesized in vitro by 9-hr-p.i. nuclei was purified, treated with a single-strand-specific endonuclease, and separated by isopycnic CsCl centrifugation. Two species with densities equivalent to viral (1.716 g/cm³) and host cell (1.702 g/cm³) DNA, respectively, were resolved. Hybridization analysis demonstrated that purified DNA, synthesized in vitro by 9-hr-p.i. nuclei, was complementary to viral DNA (38% of the input counts).     

The role of the genes imm and s in the development of immunity against T4 ghosts and exclusion of superinfecting phage in Escherichia coli infected with T4

Immunity against ghosts and exclusion of superinfecting phage were studied in cells infected with T4 phage carrying mutations in the imm (immunity), gene, the s (spackle) gene or in both. There was a strict qualitative parallelism in the development of immunity and exclusion as a function of various factors in cells receiving a specific mutant. Both imm^? and s^? T4 induced levels of immunity or exclusion close to 90% but the time pattern of appearance of these functions differed with the mutant. Cells receiving the double mutant imm^?-s^? developed very low resistance to superinfection. In all cases studied, ghost action or superinfecting phage expression followed one-hit kinetics.For a given m.o.i. of superinfection, cells infected with any of these mutants showed lower resistance (2–4×) to phage than to ghosts. There was a good correlation between levels of hydrolysis of the DNA of the superinfecting phage and those of exclusion of a genetic marker, though the amounts of marked DNA hydrolysed did not exceed 50% of the total radioactivity adsorbed. Experiments involving detachment of superinfecting phage envelopes by blending indicated preferential detachment of DNA full envelopes when the primary infecting phage contained the s^? mutation. Superinfecting ghosts inhibited formation of infective centers less than protein synthesis in cells receiving s^? T4.Experiments to test the exclusion of two genetic markers indicated that in cells infected with imm^?, s^?, or wt, expression of superinfecting genetic markers occurred through whole genome injection.The genes imm and s were not implicated in the resistance to lauryl sulphate which developed after phage infection.