A 2-year follow-up of an anti-HIV immune reaction in HIV-1 gp160-immunized healthy seronegative humans: evidence for persistent cell-mediated immunity.

The first trial of an anti-HIV immunization, using a recombinant vaccinia virus expressing gp160 (rV) for priming and paraformaldehyde-fixed rV-infected PBLs and soluble gp 160 for boosting, clearly showed an in vitro HIV-protective immune reaction. This result led us to carry out an additional 2 year Phase I clinical trial in 25 HIV-seronegative volunteers, using HIV gp 160 antigens for immunization in four different protocols. The 2 year trial showed (a) the safety of the preparations, (b) a transient humoral immunity following each boost, and (c) a long-lasting memory T-cell response. Memory cytotoxic T-lymphocytes (CTLs) induced by gp 160 antigen with or without vaccinia vector lysed HLA class I restricted target cells expressing HIV-1 env antigens. These results are consistent with CTLs being an effective component of an AIDS vaccine to control cell-to-cell viral replication, dissemination in the organism, and subsequent evolution toward AIDS.

One-year follow-up of vaccine therapy in HIV-infected immune-deficient individuals: a new strategy.

Immunization of AIDS/ARC patients with autologous cells expressing HIV antigens, although providing clinical and biological benefits, fails to restore cellular immunity. The latter result is due partly to the antiproliferative effect of HIV-1 on activated T-cells (immune suppression), which leads to blockade of specific immune reactions. To overcome immune suppression, a new vaccine strategy was designed consisting of an immunization against HIV-1 combined with components of the T-cell-suppressive (antiproliferative) network. This new vaccine treatment proved to be innocuous in mice, monkeys, and two non-HIV-infected humans. A Phase I clinical trial was performed in six patients previously under cellular immunotherapy and still presenting a cellular immune defect. Preliminary results confirmed, after a 1-year follow-up of the patients, the safety of the new vaccine, which also partially restored the cellular immune response, including anti-HIV HLA-restricted cell-mediated cytotoxicity, delayed hypersensitivity to recall antigens, and proliferation of T-cells specifically activated by recall antigens.

Biomedical applications of maghemite ferrofluid.

The use of cell-targeted ferrofluid in the characterization of modifications of cell membranes is reviewed. Maghemite ferrofluid was synthesized by the Massart method, complexed with dimercaptosuccinic acid (FF). Cell targeting by FF was developed by coupling FF to various biological effectors such as antibodies, lectins, etc, which enabled magnetic cell sorting. Modifications in erythrocyte membranes were studied using FF bound to recombinant human annexin V (AnxFF) which is very sensitive, compared to other Anx-based reagents, in the early detection of phosphatidylserine (PS) exposition on the outer leaflet of the plasma membrane. Thus PS exposition on mouse RBC was detected already after a 24-h storage at 4 degrees C and, transiently, 24 h after their infection by Plasmodium parasites, at which time the parasites are still confined to the liver, thus leading to the recruitment of young RBC and the accumulation of a species, intermediate between reticulocytes and erythrocytes, and the actual RBC target of plasmodial invasion. AnxFF revealed PS exposition on RBC from sickle cell anemia patients, following various inflammations and already after 20 days of human blood storage under blood bank conditions. Such a sensitive detection should be similar to that of macrophages which recognize exposed PS on cells and bring about the latter's elimination from the circulation. AnxFF binding determination was combined with that of cell electrophoretic mobility, glycerol resistance and filterability to characterize RBC membrane modifications in Alzheimer's disease patients which suggested a continuous damage and regeneration in RBC of these patients. A logistic analysis suggested that several three-parameter combinations could permit diagnosis of Alzheimer's disease with up to 95% accuracy. THP1 cells and macrophages, derived themselves by incubation with retinoic acid, were bound to FF and placed in a radio frequency alternating magnetic field. Magnetocytolysis was associated with FF attachment to the cells without damage to non-bound cells and without heating of the surrounding solution.

Membrane modifications of red blood cells in Alzheimer's disease.

Red blood cells (RBC) from 24 Alzheimer's disease (AD) patients, 18 age- and sex-matched nondemented (ND) patients, hospitalized in the same facility for orthopedic problems, and 18 healthy volunteers aged 30-52 years were studied in order to gain insight into the nature of RBC membrane modifications in AD. Significant differences were found between RBC from AD and ND patients or young controls respectively for annexin V-binding (45.5 +/- 18.0% vs 27.1 +/- 14.7 and 2.7 +/- 1.9, p = .003), fraction of glycerol resistant cells (30.8 +/- 11.1% vs 19.6 +/- 6.4 and 10.2 +/- 3.1, p = .026), cell electrophoretic mobility in polymer (1.028 +/- 0.022 microns sec-1 V-1 cm vs 1.046 +/- 0.022 and 1.053 +/- 0.021, p = .02) and only limited significance for the filterability (1.46 +/- 0.12 msec vs 1.58 +/- 0.11 and 1.54 +/- 0.11, p = 0.1). A logistic analysis, using simultaneously several features as independent variables, suggested the combined use of annexinV- binding, glycerol resistance, and cell filterability which allowed the assignment of 95% of patients from this cohort to the right group. A prospective analysis of a larger cohort is required for the estimation of the diagnostic value of this test battery. In addition, the high level of annexin binding is characteristic of a disruption of the phospholipid asymmetry in aged or damaged cells, while the high glycerol resistance combined with low electrophoretic mobility an rigidity characterize young RBC, thus indicating an enhanced turnover of RBC in Alzheimer's disease.

Immunogenicity of combined anti-HIV and anti-suppressive vaccine preparations.

HIV-1 antigens generate in man both a humoral and cellular immune reaction. However, in ARC/AIDS patients, the cellular response is inhibited by HIV-1 which induces an antiproliferative (suppressive) effect on activated T cells. To overcome this inhibition and up-regulate the cellular response, we designed a new vaccine strategy directed both against HIV-1 and immunosuppression and we used an immunizing preparation composed of HIV-1 antigens combined with immunoregulatory peptides prepared in a biologically inactivated but immunogenic form. In mice, this preparation induced anti-HIV-1 antibodies and a cell-mediated cytotoxicity directed against H2 restricted cells carrying HIV-1 antigens.

A low-molecular-weight RNA species in yeast mitochondria arising from a 3' end trimming of cytochrome b pre-mRNA.

Immobilization of yeast mitochondrial RNA on nitrocellulose filters without prior alkali treatment revealed a low-MW RNA species (integral of 300 bases) which hybridizes specifically to the RNA coding strand of a DNA fragment BglII-HinfI at the 3' end of the COB-BOX gene. This RNA species (often a doublet) was found in several independent preparations of wild-type mtRNA and even in box- mutants blocked in the earliest steps of mRNA maturation (e.g. box 8-1). It may, therefore, result from an endonucleolytic cut similar to that which precedes the addition of a poly-A tail in other systems.

Effect of fatty acid treatment in cerebral malaria-susceptible and nonsusceptible strains of mice.

Cerebral malaria-susceptible (C57BL/6) mice infected with Plasmodium berghei ANKA (PbA) developed low parasitemia and died from typical neurological symptoms between 8 to 10 days after infection. In contrast, nonsusceptible (BALB/c) mice developed high peripheral blood parasitemia and died 22-24 days later without neurological implications. Daily injections of fatty acids (FA) during the first 3 days after infection protected C57BL/6 mice from cerebral symptoms but had no effect on BALB/c mice. Thus, treated C57BL/6 mice developed hyperparasitemia and died 25 days after infection, like BALB/c mice. Red blood cells from C57BL/6 control mice were found to be more resistant to lysis by linoleic acid than those of BALB/c mice. Three days following infection with PbA, these differences disappeared. Treatment with FA prevented these changes. We concluded that the host's cells were altered soon after infection and that the nature and degree of alterations depended on the mouse strain, thus determining the eventual outcome of the infection. Likewise, the effects of FA might not be directed against the parasite but rather seem to act early after infection on these parasite-induced modifications of host cells.

Some yeast proteins are recognized by antibodies to reverse transcriptases from several mammalian retroviruses and display reverse transcriptase activity.

Antibodies directed against reverse transcriptases (RT) of the mammalian retroviruses SSV and RD114 recognize specifically on Western Blots yeast cytoplasmic soluble proteins of 31 and 45 kDa respectively and inhibit RT activity in this fraction. Anti RLV RTIgG recognizes a protein of 40-43 kDa in the particulate (VLP) fraction and inhibits RT activity in it. No inhibition of RT activity was seen with normal serum anti AMV RTIgG or antisera against yeast DNA polymerase I and DNA polymerase III. These yeast RTs display RNase H activity and are not inhibited by aphidicolin. They prefer Mn+2 as cofactor over Mg+2, display an optimum temperature of 25-30 degrees C and are expressed in a diploid as well as 2 haploid strains and are thus distinct from yeast Ty encoded RTs.

Isolation of Saccharomyces cerevisiae mitochondrial formyltetrahydrofolic acid:methionyl-tRNA transformylase and the hybridization of mitochondrial fMet-tRNA with mitochondrial DNA.

Formyltetrahydrofolic acid:methionyl-tRNA transformylase was isolated from Saccharomyces cerevisiae mitochondria and used to prepare yeast mitochondrial [(3)H]formylmethionyl-tRNA. This fMet-tRNA hybridizes with mitochondrial DNA but not with yeast nuclear or E. coli DNA. Unlabeled mitochondrial, but not extramitochondrial, tRNA competes in this reaction. tRNA was eluted from the hybrid and found to contain the label almost exclusively in fMet-tRNA. Yeast cytoplasmic fMet-tRNA formylated with Escherichia coli enzyme, and E. coli fMet-tRNA, do not hybridize with mitochondrial DNA. It is concluded that yeast mitochondrial tRNA(fMet) is a gene product of the mitochondrial genome.

Biochemical lesions in copper-deficient rats caused by secondary iron deficiency. Derangement of protein synthesis and impairment of energy metabolism.

Severe copper deficiency was induced in rats by rearing nursing dams and their offsprings on a semisynthetic diet comprising all the requisite nutrients and trace metals except copper. The copper-deprived rats exhibited growth retardation, severe anaemia, loss of caeruloplasmin, decrease of cytochrome oxidase, accumulation of salt-soluble collagen and a drastic decrease in iron in plasma and liver. Apart from these characteristic signs of deficiency, a marked inhibition of protein synthesis was found to occur both in vivo and in cell-free liver preparations. The curtailed ability to carry out endogenously coded amino acid incorporation into protein contrasted with the unimpaired poly(U)-acid-directed phenylalanine polymerization. This inhibition pattern, as well as the attendant disaggregation of the liver polyribosomes, suggested that the primary biosynthetic lesion was located at the stage of peptide-chain initiation. Concurrently with this alteration there was a pronounced depletion of the hepatic ATP content, associated with a parallel depression of mitochondrial respiration and an enhancement of ATPase activity. Supplementation of the copper-deficient diet with a 2-4-fold excess of iron (relative to the standard diet) prevented growth retardation and anaemia and restored normal energy metabolism, as well as unimpaired protein-synthesizing capacity. The conclusion that these disturbances were primarily determined by the secondary iron deficiency was also borne out by the finding that similar alterations occurred in rats maintained on a copper-sufficient but iron-deficient diet. On the other hand, the iron-fortified diet failed to reverse the other signs of copper deficiency, namely the loss of caeruloplasmin, the diminished rate of cytochrome oxidase and the increase of soluble collagen. The interrelations between the various biochemical lesions induced by deprivation of copper or iron are discussed and the possible role of ATP depletion in determining the derangement of protein synthesis is considered.

Yeast mitochondria contain a linear RNA strand complementary to the circular intronic bI1 RNA of cytochrome b.

bI1 RNA (excised from the first intron of the long form of the cytochrome b gene of Saccharomyces cerevisiae mitochondria) hybridizes with the two strands of a Bg/II-MboI DNA segment from this region. This fraction is resistant to digestions by DNase I and RNase T1 and disappears completely upon alkali hydrolysis. Strand-specific labeling of an intronic DNA fragment, cloned in pBR322 plasmid, was accomplished through the use of a T4 DNA polymerase. The purity of the probes was demonstrated by cloning an exon-intron fragment and labeling it by the same procedure; mRNA and pre-mRNA bands hybridized only with the transcribed DNA strand whereas bI1 RNA hybridized with the two strands under the stringent washing conditions employed (tm + 20 degrees C). Several experimental results argue against the possibility that the observation of two complementary bI1 RNA strands results from a partial self-complementarity of the RNA. A pre-mRNA intermediate from a box8 (G5046) mutant, still containing this intron, hybridizes only with the transcribed DNA strand of the pure intronic probe. The amount of the non-sense bI1 RNA strand is very low, in cells from two wild-type strains, relative to the sense RNA strand during the early stages of growth on glucose. It increases as the cells are released from glucose repression. bI1 RNA is resistant to RNase. Very little self-complementarity is seen by computer analysis of the sequence. Purified bI1 RNA is seen by electron microscopy under non-denaturing conditions as a mixture of double-stranded circular and linear molecules thus confirming the existence of the two complementary strands. The disappearance of all material following alkali hydrolysis demonstrates that these are indeed two RNA strands. Under fully denaturing conditions a mixture of single-stranded circular and linear molecules is seen as reported previously (Cell, 19, 321-329, 1980). We conclude that yeast mitochondria contain the two complementary bI1 RNA strands, one circular and the other linear. Considering a largely asymmetrical transcription of the mitochondrial genome in yeast and assuming that circularization of some intronic RNAs is part of RNA processing, we do not believe that the two strands are each a mixture of linear and circular molecules. The ratio of non-sense to sense bI1 RNA in a cytoplasmic petite mutant, A1B1, also varies according to growth conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

The significance of the pre-challenge immune status of mice for development of retrovirus-induced immunodeficiency syndrome (MAIDS).

The effects of vaccination with RNA-free viral pseudoparticles, preinfection with non-pathogenic ecotropic virus, and induction of tolerance to viral proteins in newborns on the outcome of murine immunodeficiency syndrome (MAIDS) were studied. The parameters used to follow disease progression were: lymphopenia, circulating B and T8 cells, serum IgG and IgM levels, lymphoproliferation and skin graft rejection. Immunization with RNA-free viral pseudoparticles had no effect on any of these parameters. Preinfection of adults with ecotropic virus and the induction of tolerance in newborns to virus antigens both attenuated the early symptoms of viral infection and delayed the onset of immunodeficiency and lymphoproliferation in some mice, but did not significantly alter the number of deaths due to MAIDS. Failure of immune-based therapy to produce successful protection against MAIDS suggests that immune destruction caused by the persistent virus rather than hyperimmune activity is the main pathogenic factor in this disease.

Use of annexin V-ferrofluid to enumerate erythrocytes damaged in various pathologies or during storage in vitro.

Recombinant human annexin V was bound covalently to 9 nm maghemite (gamma Fe2O3) nanoparticles, yielding annexin-ferrofluid (AnxFF), and used to separate annexin-bound red blood cells (RBC) in a magnetic field and estimate their percentage in various bloods. Annexin binding in normal human RBC increased proportionately with storage from 8% on day 2 to 42% on day 100. Enhanced AnxFF binding was associated with various pathologies. Thus, normal blood contained 10.7 +/- 5.9% AnxFF binding RBC; bloods with normal sedimentation rates (albeit with some disease necessitating analysis) contained 23.5 +/- 6.2%; those with high sedimentation rates contained 51.5 +/- 12.3%; sickle cell anaemia patients' blood contained 50.0 +/- 9.3%, and bloods from patients with other pathologies (deforming rheumatic disease, cancer necessitating chemotherapy, etc.) contained 58.6 +/- 7.6% AnxFF binding RBC. Enhanced Ca+2-dependent annexin binding reflects a loss of the asymmetric distribution of anionic phospholipids in plasma membranes which may constitute a signal for the destruction of the modified cells by the reticuloendothelial system. Once these preliminary results are confirmed, the determination of the fraction of AnxFF bound erythrocytes, following their magnetic separation, could prove a simple and rapid quality test for example in the context of blood transfusion.