Biomedical subjects
V Nussenzweig
Publications and source records attributed to V Nussenzweig.
Amastigotes of Trypanosoma cruzi sustain an infective cycle in mammalian cells.
The two main stages of development of the protozoan parasite Trypanosoma cruzi found in the vertebrate host are the trypomastigote and the amastigote. It has been generally assumed that only trypomastigotes are capable of entering cells and that amastigotes are the intracellular replicative form of the parasite. We show here that after incubation for 4 h with human monocytes in vitro 90% or more of extracellularly derived (24 h) amastigotes of T. cruzi are taken up by the cells. Within 2 h they escape the phagocytic vacuole and enter the cytoplasm, where they divide and after 4-5 d transform into trypomastigotes. Trypomastigotes also invade cultured human monocytes. However, they show a lag of several hours between invasion and the start of DNA duplication, while amastigotes commence replication without an apparent lag. Amastigotes also infect cultured fibroblasts, albeit with lower efficiency. When injected intraperitoneally into mice, amastigotes are as infective as trypomastigotes. Based on these results, and on prior findings that amastigotes are found free in the circulation of mice during the acute stage of the disease (3), it seems likely that the cellular uptake of amastigotes can initiate an alternative subcycle within the life cycle of this parasite in the mammalian host. Also, because trypomastigotes and amastigotes have diverse surface antigens, they may use different strategies to invade host cells.
Partial characterization of the cross-reacting determinant, a carbohydrate epitope shared by decay accelerating factor and the variant surface glycoprotein of the African Trypanosoma brucei.
The variant surface glycoprotein (VSG) of the African trypanosome is anchored in the cell membrane by a complex glycan attached to phosphatidylinositol. The carboxyl terminal portion of VSG contains a cryptic carbohydrate epitope, the cross-reacting determinant (CRD), that is revealed only after removal of the diacylglycerol by phosphatidylinositol-specific phospholipase C (PIPLC) or VSG lipase. Recently, we have shown that after hydrolysis by PIPLC, decay-accelerating factor (DAF)--a mammalian phosphatidylinositol-anchored protein--also contains the CRD epitope. Using a two site immunoradiometric assay in which the capturing antibody is a monoclonal antibody to DAF and the revealing antibody is anti-CRD, we now show that sugar phosphates significantly inhibited the binding of anti-CRD antibody to DAF released by PIPLC. DL-myo-inositol 1,2-cyclic phosphate was the most potent inhibitor of binding (IC50 less than 10(-8) M). Other sugar phosphates, such as alpha-D-glucose-1-phosphate, which also possess adjacent hydroxyl and phosphate moieties in cis also inhibited binding at low concentrations (IC50 = 10(-5) to 10(-4) M). In contrast, sugar phosphates which do not possess adjacent hydroxyl and phosphate moieties in cis and simple sugars weakly inhibited binding (IC50 greater than 10(-3) M). These results suggest that myo-inositol 1,2-cyclic phosphate contributes significantly to the epitope recognized by the anti-CRD antibody and is consistent with analysis of the carboxyl terminus of VSG, which also suggested the presence of the cyclic inositol phosphate. In light of the recent findings that human serum contains a glycan-phosphatidyl-inositol-specific phospholipase D, which converts DAF from a hydrophobic to a hydrophilic form lacking the CRD, the observation that the phosphate is crucial for expression of the epitope may be relevant in understanding the origin of CRD-negative DAF in urine and plasma.
Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi.
The surface of amastigotes of Trypanosoma cruzi is covered by Ssp-4, a major stage-specific glycoprotein. Ssp-4 is anchored to the cell membrane by GPI. It can be metabolically labeled with [3H]myristic acid, and is converted into a hydrophilic form by treatment with the glycan-specific phospholipase C of T. brucei, or after lysis of the parasites in non-ionic detergents. The hydrophilic form of Ssp-4 is recognized by antibodies to the cross-reactive determinant of the variant surface glycoprotein of African trypanosomes. Ssp-4 is progressively shed during the intra- or extracellular development of amastigotes preceding their transformation into epi- and trypomastigotes. We show here that T. cruzi contains a phospholipase C and that most shed Ssp-4 is hydrophilic, does not contain myristic acid, and reacts with anti-CRD. These observations provide strong evidence that phospholipase C mediates the release of this glycosyl-phosphatidylinositol-anchored protein under physiological conditions, as the parasite undergoes differentiation.
Multiple T helper cell epitopes of the circumsporozoite protein of Plasmodium berghei.
The present findings establish the lack of genetic restriction of the humoral immune response to sporozoites of Plasmodium berghei, corraborating earlier observations that mice of different strains can be protected by immunization with irradiated sporozoites. Most, if not all, anti-sporozoite antibodies are directed against the repetitive B cell epitope of the circumsporozoite (CS) protein. However, neither a peptide containing a dimer of this repeat (17.1), nor a peptide polymer containing multiple repeats induced an antibody response in mice of different H-2 and different genetic backgrounds. A yeast-derived recombinant, containing the repeat domain and part of the surrounding amino and carboxy-terminal regions of the P. berghei CS protein, induces very different levels of antibody in mice of diverse H-2 haplotypes. H-2j mice are high responders and the immunized mice are extensively protected against sporozoite challenge. The lymph node cells of the H-2j mice (but not from other strains) proliferated in the presence of peptide N, contained in the amino terminal region of the CS recombinant. Additional H-2-restricted T cell epitopes have been identified in amino and carboxy-terminal regions of the CS protein, and mice of most of the strains recognized multiple T cell epitopes. Two peptides representing T cell epitopes were synthesized in tandem with a peptide representing the B cell epitope, and were assayed for T helper activity in vivo. The antibody response of mice, primed by a single injection of sporozoites, was boosted very effectively by the administration of peptide N + 17.1 or peptide B-4 + 17.1. The B-4 T cell epitope is located in the carboxy-terminal region of the CS protein and is recognized by mice of at least four different H-2 haplotypes. These observations demonstrate that the immune response to the CS protein of P. berghei is not genetically restricted and that it contains several T cell epitopes, some of which can function as helper epitopes. In addition, they show that a synthetic sporozoite vaccine can boost the immune response to sporozoites.
Conformational restriction of peptidyl immunogens with covalent replacements for the hydrogen bond.
A new strategy for designing synthetic vaccines is presented. In this approach synthetic peptides are conformationally restricted by replacing putative hydrogen bonds with covalent mimics. The chemistry for substituting a hydrazone-ethane link (N-N = CH-CH2-CH2) for an (i + 4)----i hydrogen bond in a pentapeptide with alpha-helical potential is reported. Chemically shaping peptides to mimic the three-dimensional surfaces of proteins may enhance their immunogenicity. To test this strategy, a potential synthetic vaccine for malaria, Cys-(Asn-Pro-Asn-Ala)3-NH2, was conformationally restricted by replacing putative hydrogen bonds between asparagine side chains with a covalent replacement, an ethylene bridge, to give first generation chemically shaped immunogens. Antibodies to one of the shaped malarial peptides show a strong reaction with living Plasmodium falciparum sporozoites, a form of malaria which infects hundreds of millions of people yearly.
Decay-accelerating factor in human skin is associated with elastic fibers.
Recently a complement inhibitor, decay-accelerating factor (DAF), has been found in association with uncharacterized fibers in the extracellular matrix of human dermis. Here we show by immunohistochemistry and immunoelectronmicroscopy that DAF is on the periphery of elastic fibers, and that it appears to be associated with some microfibrillar elements that cover the fibers. That DAF is a component of these microfibrils is also suggested by studies of lesional skin from anetoderma, a disease characterized by destruction of elastic fibers. In two patients we found a network of residual fine fibers in the dermis that stain with antibodies against DAF and fibrillin (one of the proteins known to be present in the microfibrils of elastin), but do not stain with antibodies to elastin. Western blot analysis of dermal extracts with monoclonal antibodies to DAF identified a 67 kDa molecule, slightly smaller than membrane DAF, and similar in size to soluble DAF found in secretions. It is possible that together with vitronectin, an inhibitor of the membrane attack complex recently identified in association with elastin, DAF prevents damage of elastic fibers by complement.
Trypanosoma cruzi: mechanisms of cell-invasion and intracellular survival.
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Stage-specific surface antigens during the morphogenesis of Trypanosoma cruzi: developmentally regulated expression of a glycosyl-phosphatidylinositol anchored glycoprotein of amastigotes.
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Signal for attachment of a phospholipid membrane anchor in decay accelerating factor.
Decay accelerating factor (DAF) belongs to a novel group of membrane proteins anchored to the cell surface by a glycophospholipid membrane anchor that is covalently attached to the carboxyl terminus of the protein. The last 37 amino acids of membrane DAF, when fused to the carboxyl terminus of a secreted protein, are sufficient to target the fusion protein to the plasma membrane by means of a glycophospholipid anchor. This approach provides a novel means of targeting proteins to the cell-surface membrane.
Synthetic peptide vaccine confers protection against murine malaria.
A synthetic peptide, (DPPPPNPN)2D, representing a subunit of the repeat domain of the Plasmodium berghei circumsporozoite protein, was conjugated to tetanus toxoid using bisdiazobenzidine. Immunization of mice and rats with the conjugate induced high serum titers of antibodies to the parasite, and most of the animals were completely protected from malaria infection when challenged with sporozoites.
A glycan-phosphatidylinositol-specific phospholipase D in human serum.
A group of proteins anchored to the cell by phosphatidylinositol (PI) has recently been identified. The significance of this new class of membrane anchor is unknown; one possibility is that it facilitates release of the molecule by phospholipases. In fact, phospholipase C enzymes specific for the complex carboxyl-terminal glycolipids of these proteins have been isolated from African trypanosomes and from hepatocyte plasma membranes. This study reports the discovery of a glycan-PI-specific phospholipase D in human serum that cleaves both the membrane form of the variant surface glycoprotein of African trypanosomes and its glycolipid precursor, but not phosphatidylethanolamine, phosphatidylcholine, or phosphatidylinositol. Decay-accelerating factor, another PI-anchored molecule, is also cleaved by the enzyme and converted from a hydrophobic to a soluble protein. The enzyme is Ca2+-dependent, heat labile, and not affected by the inhibitor of serine proteases, phenylmethylsulfonylfluoride. Its function is not known, but the present findings indicate that it participates in the metabolism of glycolipid-anchored membrane proteins.
Interferon-gamma inhibits the intrahepatocytic development of malaria parasites in vitro.
In this study, we examined the activity of recombinant interferon (IFN)-gamma against Plasmodium berghei exoerythrocytic forms (EEF) grown in vitro within the highly differentiated human hepatoma cell line HEPG2. We assayed the effect of IFN-gamma on parasite growth by DNA hybridization using a P. berghei specific DNA probe. The specific activity of IFN-gamma against EEF is very high, and depends upon the time of lymphokine addition. When IFN-gamma is added to HEPG2 cells containing intracellular EEF, 6 hr after sporozoite invasion, parasite DNA replication is inhibited by approximately 75% at 10(3) U/ml and 50% at 1 U/ml. This treatment can either abolish or greatly reduce the infectivity of EEF for mice. When added earlier, 3 hr after completion of sporozoite invasion, IFN-gamma inhibits parasite replication to an even greater degree. The highest levels of inhibition were obtained when IFN-gamma was added 6 hr prior to sporozoite invasion (100% inhibition at 10(2) U/ml, approximately 55% inhibition at 0.1 U/ml, and 17% inhibition at 0.001 U/ml). We found that HEPG2 cells express approximately 44,000 surface receptors for IFN-gamma. These data are consistent with the view that IFN-gamma exerts its antimalarial activity by binding to surface receptors on hepatocytes and inducing intracellular changes unfavorable for parasite development. Tryptophan starvation does not appear to be involved in this process. These findings also support the idea that IFN-gamma, released from immune T cells upon encountering sporozoite antigen, may be an important effector mechanism in sterile immunity to sporozoite challenge.
Mechanism of escape of exoerythrocytic forms (EEF) of malaria parasites from the inhibitory effects of interferon-gamma.
We have studied the mechanism of inhibition by interferon-gamma (IFN-gamma) of the development of exoerythrocytic forms (EEF) of Plasmodium berghei in the livers of rats. At the time corresponding to the maximum development of EEF (44 hr after injection of sporozoites), the livers of the IFN-gamma-treated rats contained less parasite DNA as compared with controls. Twenty-four to 72 hr later, the livers of both groups of animals were free of parasites; that is, IFN-gamma treatment does not delay the development of the EEF. The decrease in parasite DNA observed in the IFN-gamma-treated rats was due to a diminution in the number, but not the size, of EEF. It appears, therefore, that treatment with the lymphokine either destroys the parasites or does not affect their replication. To study the mechanism of resistance to IFN-gamma of a small population of EEF, we subjected the parasites to four cycles of selection by IFN-gamma. The parasites from the "selected" and "nonselected" populations were equally susceptible to inhibition by IFN-gamma, indicating that the escape from IFN-gamma activity is not inherited.
A high m.w. form of decay-accelerating factor (DAF-2) exhibits size abnormalities in paroxysmal nocturnal hemoglobinuria erythrocytes.
Decay-accelerating factor (DAF) is a 70,000 Mr membrane protein that inhibits the amplification of the complement cascade on cell surfaces. Monoclonal antibodies against different epitopes of the 70,000 Mr DAF (DAF-1) recognize a second band at the position of 140,000 Mr on a Western blot of total red cell ghost proteins or partially pure DAF subjected to electrophoresis under denaturing conditions. Like DAF-1, this polypeptide (DAF-2) has the ability to accelerate decay of the C3 convertase, C4b2a, and to reincorporate into red cell membranes. A population of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack DAF-1 and also DAF-2. In addition, in some patients' red cells bearing DAF-1 of normal Mr, DAF-2 is 5,000 to 10,000 Mr smaller than normal. The structural basis for these differences in size of DAF and its PNH variants is unknown.
Expression in yeast of a Plasmodium vivax antigen of potential use in a human malaria vaccine.
DNA coding for 234 amino acids of the circumsporozoite (CS) protein of Plasmodium vivax was incorporated into yeast expression vectors. The DNA encoded all the repeat domain and codons for a highly conserved sequence, KLKQP, found in CS proteins from all malaria parasites. Yeast cells transformed with these autonomously replicating plasmids expressed, upon induction, high levels of the CS polypeptide. The malaria antigen was purified in good yields from yeast extracts and was injected into mice using alum as adjuvant. The antibodies recognized the authentic CS protein, and at high dilutions, they inhibited the invasion of hepatocytes by sporozoites in vitro.
Identification of the complement decay-accelerating factor (DAF) on epithelium and glandular cells and in body fluids.
Decay-accelerating factor (DAF) is a 70 kD membrane regulatory protein that prevents the activation of autologous complement on cell surfaces. Using immunohistochemical methods and a radioimmunometric assay based on mAbs to DAF, we found large amounts of membrane-associated DAF antigen on the epithelial surface of cornea, conjunctiva, oral and gastrointestinal mucosa, exocrine glands, renal tubules, ureter and bladder, cervical and uterine mucosa, and pleural, pericardial and synovial serosa. Additionally, we detected soluble DAF antigen in plasma, tears, saliva, and urine, as well as in synovial and cerebrospinal fluids. While plasma, tear, and saliva DAF are larger than erythrocyte (Ehu) membrane DAF by Western blot analysis, urine DAF is slightly smaller (67,000) in Mr. Unlike purified Ehu DAF, however, urine DAF is unable to incorporate into the membrane of red cells. Although its inhibitory activity on the complement enzyme C3-convertase is lower than that of Ehu DAF, it is comparable to that of serum C4 binding protein (C4bp). Biosynthetic studies using cultured foreskin epithelium and Hela cells disclosed DAF levels (approximately 2 X 10(5) molecules/cell) exceeding those on blood cells. In addition, these studies revealed the synthesis of two DAF species, one with apparent Mr corresponding to that of epithelial cell membrane DAF and the other to urine DAF, suggesting that the urine DAF variant arises from adjacent epithelium. The function of DAF in body fluids is unknown, but the observation that urine DAF has C4bp-(or factor H-)like activity shows that it could inhibit the fluid phase activation of the cascade.
Isolation of decay accelerating factor (DAF) by a two-step procedure and determination of its N-terminal sequence.
Decay-accelerating factor (DAF) from human red cell membranes was purified by a two-step procedure involving anion exchange and immunoaffinity chromatography. The DAF preparations were purified to homogeneity as judged by silver staining. In several experiments, the final product yields were approximately 23% of the total DAF present in the initial membrane extracts. The purified DAF retained its ability to inhibit the classical pathway C3-convertase and to reincorporate into cell membranes. An amino-terminal sequence was obtained by gas-phase sequencing. Rabbit antibodies to a synthetic peptide representing part of this sequence reacted with purified reduced membrane DAF by Western blotting and by a solid-phase immunoradiometric assay.