Antiapoptotic activity of the Bowman-Birk inhibitor can be attributed to copurified phospholipids.

Previous studies have shown that extracts from soy possess potent antiapoptotic activity in in vitro and in vivo models. We recently reported that this antiapoptotic activity can be attributed to the presence of specific phospholipids. In this study, a conventional preparation of the soy-derived Bowman-Birk inhibitor (BBI) was tested for antiapoptotic activity in a C3H/10T1/2 cell serum deprivation assay. The BBI preparation was separated into lipid- or protein-containing fractions by organic extraction. The lipid fraction contained only antiapoptotic activity; the protein fraction contained only enzyme inhibition activity. We therefore conclude that the antiapoptotic activity of the BBI preparation is due to specific phospholipids that copurify with BBI. These phospholipids retain their antiapoptotic activity after autoclave treatment, whereas autoclave treatment of the protein fraction results in a loss of its enzyme inhibition activity.

Soy-derived antiapoptotic fractions protect gastrointestinal epithelium from damage caused by methotrexate treatment in the rat.

The ability of a previously described soy-derived antiapoptotic fraction (SDAAF), a soy water extract (Lexirin), and raw soy flour to inhibit methotrexate (MTX)-induced gastrointestinal damage was evaluated by histological examination of duodenal/jejunal sections from MTX-treated rats. Male Sprague-Dawley rats were fed diets containing casein as a sole protein source or diets supplemented with fractions isolated from soy (SDAAF or Lexirin) before and after MTX treatment. The soy fractions were also shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in mouse embryonic C3H10T1/2 cells. Protein sequence (Lexirin) and enzyme activity (Lexirin and SDAAF) were also analyzed. Rats that received SDAAF- and Lexirin-supplemented diets had significantly reduced necrotic and apoptotic damage in the duodenal mucosa, as demonstrated by difference in villi height, mitotic activity, epithelial cell height, and inflammatory response.

Characterization of a recombinant Onchocerca volvulus antigen (Ov33) produced in yeast.

A yeast (Saccharomyces cerevisiae) expression system has been adapted to produce reagent quantities of a major Onchocerca antigen, Ov33. Using a pool of monoclonal antibodies produced against third-stage larvae, a cDNA library constructed from adult O. volvulus worms was screened. Twenty-seven cDNAs were isolated, two of which had sequence homology to Ov33, a putative aspartyl protease inhibitor, which is the immunodominant antigen of O. volvulus. These cDNAs were expressed at high levels intracellularly or through the secretory pathway of S. cerevisiae. Localization studies using antisera produced against purified recombinant protein demonstrated that Ov33 is a very abundant parasite protein present in the hypodermis, muscle, and uterus of female worms, as well as in embryonic microfilariae. The soluble recombinant protein secreted by yeast (C71) demonstrated inhibitory activity against the aspartyl protease pepsin. Antibodies to the recombinant protein-mediated leukocyte adherence to and killing of skin microfilariae. The sensitivity of a diagnostic test using recombinant Ov33 was evaluated using sera from 441 patients. The mean sensitivities for the two recombinant constructs, C27 and C71, were 82.2% and 85.4%, respectively. The combined sensitivity using both recombinant proteins was 94%.

Fine epitope specificity of antibodies to region II of the Plasmodium vivax circumsporozoite protein correlates with ability to bind recombinant protein and sporozoites.

Recent work has suggested that important B- and T-cell epitopes on the circumsporozoite protein (CSP) of Plasmodium vivax lie external to the major repeat regions of the protein. We have studied two naturally exposed human populations (Caucasian and Papua New Guineans) and determined the antibody response to yeast-derived recombinant CSPs, overlapping synthetic peptides spanning amino acids 76 348 of the Belem P. vivax CSP and overlapping peptides representing the variant repeats of the VK247 strain of P. vivax. We have demonstrated that the P. vivax CSP-specific antibody response is directed towards areas within the repeat region as well as areas external to this; but the dominant epitopes recognized by the two populations studied, were distinct. One epitope, lying external to the repeats and recognized by both populations, partially overlaps an area of the protein referred to as region II-plus. Sera from malaria-exposed Papua New Guineans and Thais contained antibodies to this epitope (V22, single letter amino acid sequence TCGVGVRVRRRVNAANKKPE) which were capable of recognizing sporozoites, as determined by quantitative inhibition IFA. Seventeen percent of PNG sera had antibodies to this peptide compared with 33% who had antibodies to the central repeats of the protein. Immunization of mice with recombinant CSP did not induce antibodies to V22. However, immunization with overlapping peptide epitopes representing this region (V21 or V22) induced specific antibodies but only two sera recognized both V21 and V22 and, by inference, the overlapping peptide sequence (TCGVGVRVRR). Antibodies in these two sera could bind recombinant CSP in ELISA; however, in contrast, nine sera which recognized either V21 or V22 alone did not bind CSP. Only one of two sera containing antibodies recognizing CSP stained P. vivax sporozoites. This serum also recognized an epitope dependent upon two amino acids aminoterminal to V22. These data suggest that the fine specificity of antibodies is a critical determinant for binding to both recCSP and sporozoites.

Antiapoptotic compound to enhance hypothermic liver preservation.

BACKGROUND: Apoptosis (programmed cell death) occurs as a consequence of global organ ischemia during isolation and storage prior to transplantation. If apoptosis is inhibited during ischemia, organ preservation should be improved, and the length of time for permissible storage may be increased. The objective of this study was to test the effect of a newly developed antiapoptotic compound, LXR-015, during extended hypothermic liver preservation. METHODS: Three groups of 12 rats each were studied. In the normal group, liver function was studied immediately after harvesting. In the study group, harvested livers were flushed with Euro-Collins solution (30 ml/kg body weight) containing LXR-015 at a concentration equivalent to 9 mg/kg animal body weight (300 microg/ml). The livers were then stored at 4 degrees C for 24 hr before liver function was studied. In the control group, harvested livers were flushed with Euro-Collins solution without LXR-015 and then stored at 4 degrees C for 24 hr before liver function was studied. RESULTS: Portal venous flow was higher (P<0.05) in the normal and study groups compared with the control group. Portal venous resistance was lower (P<0.05) in the normal and study groups compared with the control group. Liver tissue oxygen consumption in the study group was significantly higher than in both the normal and control groups (P<0.05). Liver enzyme production (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase) was higher in the control group than in either the study or normal group (P<0.05). Bile production in both the normal and study groups was higher than in the control group (P<0.05). The liver tissue wet to dry weight ratio in both the normal and study groups was lower than in the control group (P<0.05). Histopathology studies revealed fewer apoptotic bodies (P<0.05) in both the normal (1.70+/-0.15 per high-power field) and study groups (2.08+/-0.10 per high-power field) than in the control group (7.92+/-.33 per high-power field). CONCLUSIONS: Adding an antiapoptotic compound, LXR-015, to Euro-Collins solution significantly improves hypothermic preservation of the rat liver compared with Euro-Collins solution alone.

A soy-derived antiapoptotic fraction decreases methotrexate toxicity in the gastrointestinal tract of the rat.

The ability of a soy-derived antiapoptotic fraction to inhibit methotrexate-induced gastrointestinal toxicity was examined. Male Sprague-Dawley rats treated with methotrexate were fed diets containing casein as a sole protein source or diets supplemented with a protein-phospholipid fraction isolated from soy flour. This soy fraction has also been shown to inhibit serum deprivation-induced programmed cell death (apoptosis) in the mouse embryonic C3H10T1/2 cell. Rats that received high doses of the soy-derived antiapoptotic fraction-supplemented diets experienced significantly less weight loss and diarrhea and better maintained their pretreatment appetite.

Prevention of rat neonatal cardiomyocyte apoptosis induced by simulated in vitro ischemia and reperfusion.

Apoptosis, or programmed cell death, is an active metabolic response to physiological signals or exposure to cytotoxic agents. Recent evidence has shown that the cell death response can be modified by agents presumed to be unrelated to the initial signal, but capable of interfering with the molecular mechanisms of the apoptotic pathway progression. Here we show the results of investigations on the use of a phospholipid-based pharmaceutical preparation for suppression of myocardial damage. First, we show that serum or serum/glucose deprivation, in vitro ischemia with subsequent simulated reperfusion, inhibition of protein synthesis, and treatment with ceramide, staurosporine, adriamycin, cis-platinum and menadione induce apoptotic death in a primary culture of rat neonatal cardiomyocytes. Then we demonstrate that a mixture of specific phospholipids, which has been originally purified from soy flour on the basis of its anti-apoptotic activity, prevents cardiomyocyte death induced by serum or serum/glucose deprivation, by ischemia with subsequent simulated reperfusion, and by ceramide, but not by other cytotoxic treatments. This suggests that ceramide, a lipid secondary messenger which triggers apoptosis induced by some cytotoxic agents, may be involved in the process of signaling ischemia/reperfusion induced apoptotic death of cardiomyocytes. These results further demonstrate that an active pharmaceutical preparation for the suppression of cardiomyocyte death can be formulated based upon a novel strategy of apoptosis modification.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes.

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

Onchocerca volvulus: identification and characterization of an immunogenic eggshell protein (Oveg1).

A major antigen recognized by human sera in Onchocerca volvulus infections is a parasite eggshell protein. The cDNA clone for this antigen was isolated from a lambda gt11 O. volvulus cDNA library using antisera from patients with high microfilarial counts. Sequence analysis of the cDNA clone predicts a polyglutamine repeat near the 5' end of the cDNA, and a motif of four arginines near the 3' end, reminiscent of that found in many regulatory proteins. The cDNA was subcloned into a yeast expression vector and reagent quantities of recombinant antigen produced in Saccharomyces cerevisiae. Antisera produced to the recombinant purified protein localized the antigen to the eggshell of developing microfilariae within the adult female uterus. No other sites of Oveg1 expression were noted in adult worms, but labeling was seen in internal membrane structures of L3 larvae. Sera from infected chimps recognized Oveg1 only after infections became patent. Sera from infected humans showed reactivity to Oveg1 that varied from 39 to 95%, depending upon the geographic location.

Immunogenic properties of the M. leprae recombinant 18-kDa antigen purified from Saccharomyces cerevisiae; enhancement of delayed-type hypersensitivity after gamma-irradiation.

In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described [Biotech. Lett. 16 (1994) 1241-1246]. The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100). The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice. Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained. Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response. The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M. leprae infection.

Saccharomyces cerevisiae recombinant Pfs25 adsorbed to alum elicits antibodies that block transmission of Plasmodium falciparum.

Antibodies to Pfs25, a cysteine-rich 25-kDa protein present on the surface of Plasmodium falciparum zygotes, can completely block the transmission of malaria parasites when mixed with infectious blood and fed to mosquitoes through a membrane feeding apparatus. Recently, a polypeptide analog, Pfs25-B, secreted from recombinant Saccharomyces cerevisiae was found to react with conformation-dependent, transmission-blocking monoclonal antibodies and to elicit transmission-blocking antibodies in experimental animals when emulsified in either Freund's or muramyl tripeptide adjuvant. In this study, Pfs25-B adsorbed to alum induced transmission-blocking antibodies in both rodents and primates. Bacterially produced Pfs25, however, did not elicit complete transmission-blocking antibodies in rodents. Furthermore, unlike monoclonal antibodies to Pfs25, which block transmission only after ookinete development, antisera to Pfs25-B adsorbed to alum appeared to block the in vivo development of zygotes to ookinetes as well.

Life-spans of human T-cell responses to determinants from the circumsporozoite proteins of Plasmodium falciparum and Plasmodium vivax.

The longevity of specific human memory T-cell responses is largely unknown. However, a knowledge of the duration of memory is important for understanding immunity to an organism and for planning vaccine intervention. To address this, we have examined T-cell memory to malaria by determining T-cell responses by subjects recently exposed to peptides spanning the circumsporozoite (CS) proteins of two species of malaria-causing organisms, Plasmodium falciparum and Plasmodium vivax. Responses to vivax CS peptides by exposed Thai subjects were more frequent than responses by nonexposed individuals, permitting identification of determinants seen by vivax-induced responses. At the population level, there appears to be life-long memory, as the time since individuals were exposed did not diminish responsiveness to these determinants. In contrast, falciparum-exposed subjects were largely indistinguishable from nonexposed controls in responsiveness to falciparum CS determinants. However, a single peptide (F16: DNEKLRKPKHKKLKQPGDGN) was recognized significantly more frequently by P. falciparum-exposed than nonexposed Thai subjects. T cells responsive to this peptide were CD450+ and produced gamma-interferon. In contrast to the response to the vivax determinants and the other falciparum determinants, responsiveness to F16 was undetectable or minimal 2 years after exposure. Our data provide the average life-spans of certain malaria-specific T cells and are consistent with, but do not prove, the hypothesis that antigenic persistence (in the form of P. vivax hypnozoites) correlates with persistence of human T-cell memory.

Epitopes recognized by HIV-SF2 nef-specific CD4+ T-cell clones generated from HIV-1-uninfected donors.

Human T-cell clones with specificity to the HIV-1 nef protein were generated by the in vitro stimulation of peripheral blood mononuclear cells (PBMCs) from HIV-1-seronegative donors with purified nef from the HIV-SF2 isolate produced in genetically engineered yeast. Here the characterization is described of a total of seven discrete clones derived from five different donors. Each clone was CD3+ CD4+ CD8- as determined by FACS analysis. The epitopes recognized by these clones were identified using synthetic overlapping peptides spanning the entire length of nef. Six discrete helper T-cell epitopes located in five distinct regions of nef were identified by this approach. Three of these epitopes are more than 80% conserved among all HIV-1 nef proteins for which sequence data are available. The remaining epitopes are in regions of nef that vary among isolates. Many of the epitopes recognized by our clones overlap T-cell epitopes identified by others examining T-cell responses to nef in HIV-1-infected patients and immunized animals. Using partially class II-matched EBV-transformed B-cell lines, we were able to identify five different HLA class II alleles which encode restricting elements for the in vitro nef-specific proliferative response of these clones (DR1, DRw15(2), DRw6, DQw7, DP5).

Protein expression in yeast as an approach to production of recombinant malaria antigens.

The selection of a system suitable for expression of recombinant malaria antigens for vaccine development is, in the final analysis, empirical. However, experience gained with both malaria antigens and other recombinant proteins has provided helpful guidelines. Recombinant DNA technology has been successfully applied to the development of vaccines against a number of human diseases. For example, recombinant DNA-derived hepatitis B virus surface antigen has been produced from both prokaryotic and eukaryotic systems. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics, and vaccine production. Both intracellular and secretory systems have been developed and optimized for the production of high levels of recombinant proteins. Recombinant DNA technology, and in particular yeast expression systems, have been successfully used to produce malaria antigens, several of which have been protective in various animal models. In contrast, attempts to produce sufficient quantities of antigens for a malaria vaccine from in vitro cultures of the malaria parasite have been unsuccessful. Recombinant proteins can be produced and purified from yeast in large quantities and at low cost, each being requirements for a vaccine to be used in a global vaccination program against malaria.

Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity.

We report the results of vaccination trial 2 of Panamanian Aotus monkeys with a recombinant blood-stage antigen, SERA 1, of the malaria parasite Plasmodium falciparum. Monkeys were immunized with SERA 1, a 262-amino-acid fragment (amino acids 24 to 285) of the 989-amino-acid SERA protein produced by the Honduras 1 strain of the parasite. Immunization mixtures contained 100 micrograms of recombinant SERA 1 protein per dose mixed with one of five different adjuvants. The protein mixed with either Freund's adjuvant or MF75.2 adjuvant stimulated protective immunity. When other P. falciparum antigens were included in the SERA 1-Freund's adjuvant mixture, no protective immunity was observed, although high anti-SERA 1 antibody titers were produced. Three other adjuvants mixed with SERA 1 failed to induce a protective immune response. These results, their relationship to those reported previously in the first vaccination trial (trial 1), and their relationships to the quantitative measurement of anti-SERA 1 antibodies in enzyme-linked immunosorbent assays provided insights into the induction of a protective immune response in vaccinated monkeys.

Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: further studies using SERA 1 and MF75.2 adjuvant.

We describe the third of three vaccination trials of Panamanian Aotus monkeys with a recombinant blood-stage antigen derived from the malaria parasite Plasmodium falciparum. Immunization was performed with an N-terminal region of the SERA antigen (serine repeat antigen protein), SERA 1, that contains a 262-amino-acid fragment including amino acids 24 to 285 of the 989-amino-acid SERA protein. Vaccinations were carried out with the recombinant protein mixed with either Freund's, MF75.2, or MF59.2 adjuvant. A control group that did not receive SERA 1 but only MF75.2 adjuvant was included. Monkeys vaccinated with the antigen MF59.2 mixture produced low anti-SERA 1 titers and were not protected. Monkeys vaccinated with antigen and Freund's adjuvant had, in general, a higher average anti-SERA 1 titer (107,278) than did monkeys immunized with SERA 1 and MF75.2 (40, 143), yet monkeys in both groups were well protected. Monkeys that received only MF75.2 developed neither detectable anti-SERA 1 nor anti-P. falciparum antibodies prior to or 10 days after parasite challenge, yet were apparently protected against infection. Monkeys vaccinated with either SERA 1 and Freund's, SERA 1 and MF75.2, or MF75.2 alone and that had been challenged but did not develop a countable parasitemia were treated with a curative dose of mefloquine 100 days after parasite challenge and then rechallenged 40 days later. None of the five rechallenged monkeys that had originally received SERA 1 and Freund's developed a countable parasitemia. Only one of five rechallenged monkeys that originally received SERA 1 and MF75.2 developed a high countable parasitemia, while two animals developed a barely countable parasitemia. Four of the rechallenged monkeys that had originally received only MF75.2 developed a moderate to high countable parasitemia. The results indicate that vaccination with SERA 1 and either Freund's or MF75.2 adjuvant provides protection and vaccination with MF75.2 alone can provide a temporary protection unrelated to the induction of anti-SERA 1 or antimalarial antibodies.

An experimental vaccine cocktail for Plasmodium falciparum malaria.

Surface proteins from several different life-cycle stages of the malaria parasite Plasmodium falciparum were expressed at high levels in the yeast Saccharomyces cerevisiae. Purified proteins, both individually and in cocktails, were used to immunize mice and goats in conjunction with either Freund's adjuvant or a muramyl tripeptide-based adjuvant. Immune responses were measured by enzyme-linked immunosorbent assays and by the ability of antisera to inhibit (1) the invasion of hepatocytes by live sporozoites, (2) in vitro invasion of human erythrocytes by live merozoites, and (3) the development of oocytes in the mosquito vector. These results suggest that cocktails of different stage-specific antigens can provide the components necessary to block the development of the malaria parasite at multiple stages of its life cycle.

Malaria transmission-blocking vaccines.

Antibodies to surface proteins of the sexual stages of Plasmodium falciparum block completely the transmission of these malaria parasites. Transmission-blocking vaccines therefore represent a powerful and novel approach to controlling the spread of this lethal disease.

The predicted proteinase furin is not the hepatic proalbumin convertase.

Rat and chicken liver microsomal membranes were used to investigate the relationship between proalbumin processing activity and the predicted proteinase furin. Two polyclonal antisera directed against the predicted catalytic domain of furin showed the highest level of immunoreactivity in a microsomal fraction that had minimal proalbumin converting activity. Extracts of the fraction containing most converting activity lacked detectable furin. In addition, the proalbumin convertase was not inhibited by the anti-furin antisera. These results strongly suggest that furin is not responsible for the in vivo cleavage of proalbumin.

Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T).

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.