CD8+ lymphocyte depletion without SIV infection does not produce metabolic changes or pathological abnormalities in the rhesus macaque brain.

BACKGROUND: Simian immunodeficiency virus (SIV) infection and persistent CD8(+) lymphocyte depletion rapidly leads to encephalitis and neuronal injury. The objective of this study is to confirm that CD8 depletion alone does not induce brain lesions in the absence of SIV infection. METHODS: Four rhesus macaques were monitored by proton magnetic resonance spectroscopy ((1) H-MRS) before and biweekly after anti-CD8 antibody treatment for 8 weeks and compared with four SIV-infected animals. Post-mortem immunohistochemistry was performed on these eight animals and compared with six uninfected, non-CD8-depleted controls. RESULTS: CD8-depleted animals showed stable metabolite levels and revealed no neuronal injury, astrogliosis or microglial activation in contrast to SIV-infected animals. CONCLUSIONS: Alterations observed in MRS and lesions in this accelerated model of neuroAIDS result from unrestricted viral expansion in the setting of immunodeficiency rather than from CD8(+) lymphocyte depletion alone.

Changes in MRS neuronal markers and T cell phenotypes observed during early HIV infection.

OBJECTIVE: To determine if changes in brain metabolites are observed during early HIV infection and correlate these changes with immunologic alterations. METHODS: Eight subjects with early HIV infection, 9 HIV-seronegative controls, and 10 chronically HIV-infected subjects without neurologic impairment underwent 1H magnetic resonance spectroscopy. Subjects with early stage infection were identified near the time of HIV seroconversion and imaged within 60 days of an evolving Western blot, while still having detectable plasma virus. Subjects had blood drawn for viral RNA and T cell quantification. RESULTS: Both N-acetylaspartate (NAA) and Glx (glutamate + glutamine) were decreased in the frontal cortical gray matter of seropositive subjects. NAA levels were found to be decreased in the centrum semiovale white matter of chronically HIV-infected subjects, but not in those with early infection. Both HIV-infected cohorts demonstrated a lower number of CD4+ T lymphocytes and a higher number of CD8+ T lymphocytes in their blood. Lower NAA levels in the frontal cortex of subjects with early infection were associated with an expansion of CD8+ T cells, especially effector CD8+ T cells. CONCLUSIONS: These results verify metabolism changes occurring in the brain early during HIV infection. Lower NAA and Glx levels in the cortical gray matter suggests that HIV causes neuronal dysfunction soon after infection, which correlates to the expansion of CD8+ T cells, specifically to an activated phenotype. Utilizing magnetic resonance spectroscopy to track NAA levels may provide important information on brain metabolic health while allowing better understanding of the virus-host interactions involved in CNS functional deficits.

The role of therapeutic apheresis in the treatment of cancer: a review.

Immunosuppression is a hallmark of advanced malignancies in man. Over the past 40 years, many investigators have identified soluble immunosuppressive factors in blood, serum, ascitic fluid, and pleural fluid from cancers in man and other species. Suppressive factors have also been identified that are produced by tumors. The description of immunosuppressive factors in the blood of vertebrates who either have cancer or who are pregnant is significant, for only in pregnancy and cancer does a seemingly normal immune system tolerate immunogenic neoantigen. Tumor necrosis factors (TNFs) are known to be pleiotropic cytotoxic cytokines that are produced by macrophages and lymphocytes. These cells are thought to be suppressed in patients who have cancer or who are pregnant. Recently, elevated blood levels of soluble tumor necrosis factor receptors (sTNFRs) have been reported in the blood in a variety of cancers and pregnancy. In 1990, after our initial publication of the discovery of sTNFRs in the serum and low molecular weight ultrafiltrates of serum from a variety of cancer patients, others confirmed significant elevations of sTNFRs in cancer patients. This elevation was found to correlate with a poor prognosis. The biologic activity of proinflammatory cytokines as well as the suppressive role of their shed receptors is herein reviewed. Work with cancer patients using ultrapheresis to reduce these suppressive molecules by the authors and others is reviewed. Several recommendations are made for future directions.

Modulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein.

E1 is the DNA replication origin recognition protein for bovine papillomavirus (BPV), and it carries out enzymatic functions required for initiation of viral DNA replication. Cellular mechanisms likely play a role in regulating BPV DNA replication. We are investigating the role of phosphorylation of E1 on viral replication in vivo and on E1 activity in vitro. Serine 109 is a phosphoacceptor in vivo and is targeted by protein kinase A and protein kinase C in vitro. A viral genome carrying a serine 109 to alanine mutation replicates more efficiently than wild-type in vivo in a transient replication assay. Furthermore, purified mutant protein, while having wild-type levels of ATPase activity, is able to bind more origin-containing DNA than wild-type E1. Phosphorylation therefore appears to play a selective role in modulating a specific E1 function during viral DNA replication.

Expression of poliovirus P3 proteins using a recombinant vaccinia virus results in proteolytically active 3CD precursor protein without further processing to 3Cpro and 3Dpol.

The expression of the poliovirus genome occurs by the translation of a single open reading frame to generate a long polyprotein which is subsequently processed by viral encoded proteases. The initial proteolytic cleavages result in the production of a P1 polyprotein which contains the capsid proteins, and the P2 and P3 polyproteins which contain proteins required for replication. The P3 polyprotein consists of the 3AB protein (containing the viral genome-linked protein, VPg), the viral protease, 3Cpro, and RNA polymerase, 3Dpol. To further study the expression and proteolytic processing of poliovirus P3 proteins in vivo, we have utilized recombinant vaccinia virus vectors to express nucleotides 5240-7400 containing the P3 region proteins of poliovirus. The P3 protein expressed from the recombinant vaccinia virus VV-P3 exhibited in vivo proteolytic activity as evident by processing of the polyprotein to generate the 3CD protein, consisting of a fusion between the 3Cpro and 3Dpol proteins. Further processing of the 3CD protein to 3Cpro and 3Dpol, however, was not detected in cells infected with VV-P3. Subcellular fractionation of VV-P3-infected cells demonstrated that the 3CD protein was present in both the soluble and membrane fractions. Finally, the 3CD protein expressed from VV-P3 was stable in cells co-infected with VV-P3 and poliovirus and no further processing to 3Dpol was detected. These results are discussed with regards to in vivo studies which suggest that the 3CD polyprotein is not a precursor to 3Dpol in poliovirus-infected cells.

The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.

Bovine papillomavirus (BPV) DNA replication occurs in the nucleus of infected cells. Most enzymatic activities are carried out by host cell proteins, with the viral E1 and E2 proteins required for the assembly of an initiation complex at the replication origin. In latently infected cells, viral DNA replication occurs in synchrony with the host cell chromosomes, maintaining a constant average copy number of BPV genomes per infected cell. By analyzing a series of mutants of the amino-terminal region of the E1 protein, we have identified the signal for transport of this protein to the cell nucleus. The E1 nuclear transport motif is highly conserved in the animal and human papillomaviruses and is encoded in a similar region in the related E1 genes. The signal is extended relative to the simple nuclear localization signals and contains two short amino acid sequences which contribute to nuclear transport, located between amino acids 85 and 108 of the BPV-1 E1 protein. Mutations in either basic region reduce nuclear transport of E1 protein and interfere with viral DNA replication. Mutations in both sequences simultaneously prevent any observable accumulation of the protein and reduce replication in transient assays to barely detectable levels. Surprisingly, these mutations had no effect on the ability of viral genomes to morphologically transform cells, although the plasmid DNA in the transformed cells was maintained at a very low copy number. Between these two basic amino acid blocks in the nuclear transport signal, at threonine 102, is a putative site for phosphorylation by the cell cycle regulated kinase p34cdc2. Utilizing an E1 protein purified from either a baculovirus vector system or Escherichia coli, we have shown that the E1 protein is a substrate for this kinase. An E1 gene mutant at threonine 102 encodes for a protein which is no longer a substrate for the p34cdc2 kinase. Mutation of this threonine to isoleucine had no observable effect on either nuclear localization of E1 or DNA replication of the intact viral genome.

The phylogeny of oncology.

A review of reproductive biology and oncologic immunology reveals striking similarities between the tolerance of neoantigen as demonstrated in pregnancy and cancer. The author discusses the phylogeny of sexual reproduction and the oncologic condition, and suggests that an evolved mechanism of acquired tolerance to HLA-incompatible tissue necessitated by sexual reproduction consequently provides a mechanism for the tolerance of cancer.

Apheresis of low molecular weight protein fraction and the onset of labor.

In our study, we subjected six (6) pregnant goats of known gestational age to whole blood ultrapheresis using an extracorporeal circuit. The serum fraction removed was replaced with equal volume of the same material prepared from non-gravid goats. The serum fraction removed was identified as to content of clinically measurable parameters and plotted as permeability coefficient compared with molecular weight. The goats were observed to enter the first stage of labor as a consequence of this form of apheresis and to deliver normal products of conception. No clinically significant deleterious effects on the goats were observed. There was no evidence of complement activation, nonspecific heparin effect, or endotoxin effect. We consider the possibility that soluble mediators of immune suppression may play a role in the maintenance of pregnancy and that the onset of the first stage of labor may be an immunologic event.

Continuous whole blood UltraPheresis procedure in patients with metastatic cancer.

Sixteen patients with metastatic cancer, each with bidirectionally measurable disease, were treated with a total of 24 membrane UltraPheresis procedures each to remove a low molecular weight (less than 150,000 daltons) plasma fraction. No other oncologic treatment was applied during the 2 months of study. The procedure was generally well tolerated, and no clinically significant adverse effects were observed from the procedure. A consistent tumor-specific inflammatory response was observed following the UltraPheresis procedure and was associated with lymphocytic infiltration of tumor and tumor necrosis that was demonstrated in those patients evaluable by repeat biopsy. In some patients, anergy was reversed and Karnofsky status improved. Six of the 16 patients had reduction of the sum of mean cross-sectional diameters of measureable lesions by 50% or more.

Expression of enzymatically active poliovirus RNA-dependent RNA polymerase in Escherichia coli.

The poliovirus genome is replicated by a virus-encoded RNA-dependent RNA polymerase (RNA polymerase). The RNA polymerase is first synthesized as a larger precursor polypeptide, which is subsequently processed by a viral proteinase, 3Cpro, to give the mature polymerase molecule, 3Dpol. To further characterize the poliovirus RNA polymerase, we have constructed plasmids that expressed this protein in Escherichia coli. The plasmids consisted of fusions between the E. coli DNA encoding the first 13 amino acids of the trp operon leader protein and viral genes encoding the 3Cpro and 3Dpol polypeptides. E. coli harboring such plasmids gave significant, inducible levels of enzymatically active RNA polymerase as determined by the poly(A).oligo(U) poly(U) polymerase assay. The purified RNA polymerase activity from E. coli corresponded to a protein with the approximate molecular weight of the mature 3Dpol protein. The availability of a recombinant, enzymatically active poliovirus RNA polymerase provides a system in which we can precisely delineate the role this enzyme plays in the regulation of poliovirus replication.

Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism.

Different isolates of influenza virus show a high degree of amino acid sequence variation in their surface glycoproteins. Conserved residues located in the substrate-binding pocket of the influenza virus neuraminidase are therefore likely to be involved in substrate binding or enzyme catalysis. In order to study the structure and function of the active site of this protein, a full-length cDNA clone of the neuraminidase gene from influenza A/Tokyo/3/67 was subcloned into aN M13 vector and amino acid substitutions were made in selected residues by using the oligonucleotide mismatch technique. The mutant neuraminidase genes were expressed from a recombinant SV40 vector, and the proteins were analyzed for synthesis, transport to the cell surface, and proper three-dimensional folding by internal and surface immunofluorescence. The mutant neuraminidase proteins were then assayed to determine the effect of the amino acid substitution on enzyme activity. Twelve of the 14 mutant proteins were correctly folded and were transported to the cell surface in a manner identical with that of the wild type. Two of these have full enzyme activity, but seven mutants, despite correct three-dimensional structure, have completely lost neuraminidase activity. Two mutants were active at low pH. The properties of the mutant enzymes suggest a possible mechanism of neuraminidase action.

[Expression and mutagenesis of genes coding for protein synthesis in the influenza virus]. / Issledovanie ékspressii i mutageneza genov, kodiruiushchikh sintez belkov virusa grippa.

Double-stranded cDNA copies of the neuraminidase genes of influenza viruses A/Tokyo/3/67 (N2), A/tern/Australia/G70C/75 (N9), and B/Lee/40, and the hemagglutinin genes of A/Memphis/1/71 (H3) and B/Hong Kong/8/73 were cloned into a SV40 vector in which the late region was replaced by the influenza sequences. Thus the influenza genes were expressed in transfected cells under the control of the SV40 late promoter. The Tokyo/67 neuraminidase gene was modified by oligonucleotide-directed site-specific in vitro mutagenesis. Several of the amino acid residues which are conserved in all known neuraminidases and which line the sialic acid binding pocket were changed, and the mutant gene ligated back into the SV40 vector. Five mutations which have been fully characterized resulted in synthesis of a protein which had totally lost neuraminidase enzyme activity.

How antibodies recognize virus proteins.

Numerous studies have addressed the nature of antibody-antigen interaction, but only recently have three-dimensional structures of complexes of antibodies with protein antigens been reported, one with lysozyme and one with the influenza virus antigen neuraminidase. Both structures show that there are epitopes involving about 16 amino acids on surface loops of the antigen. In the lysozyme complex the interaction of the components is rigid, but there is a degree of structural flexibility in the formation of the complex with neuraminidase. In this article, Peter Colman, Graeme Laver and their colleagues discuss some speculative implications of the results currently available.

Loss of enzyme activity in a site-directed mutant of influenza neuraminidase compared to expressed wild-type protein.

Full-length double-stranded DNA copies of the neuraminidase (NA) gene of influenza virus A/Tokyo/3/67 (N2) and a mutant generated in vitro by site-specific, oligonucleotide-directed mutagenesis with a substitution of leucine for tryptophan at position 178 were cloned into an SV40 late replacement expression vector. Indirect immunofluorescence of cells infected with these recombinant vectors showed the presence of NA protein in the cytoplasm and on the surface of infected cells. Cells expressing the wild-type protein showed neuraminidase enzyme activity for both fetuin, a sialated glycoprotein (mol wt = 50,000) and N-acetylneuraminyl lactose, a trisaccharide (mol wt = 600). This enzyme activity was inhibited by 44% toward N-acetylneuraminyl lactose and by 98% toward fetuin by adding anti-NA antibody before substrate. In contrast, cells expressing the mutant NA had no detectable enzyme activity for either substrate. The conserved nature of the tryptophan at position 178 in all known NA strains, its location in the substrate binding pocket in the three-dimensional structure and the lack of activity of the mutant protein indicate that this residue is essential for enzyme activity.

Sequence of the neuraminidase gene of influenza virus A/Tokyo/3/67 and previously uncharacterized monoclonal variants.

A full-length cDNA copy of the neuraminidase (NA) gene of influenza strain A/Tokyo/3/67 was cloned into the plasmid pBR322, and the nucleotide sequence of the gene was determined. In addition, the sequence changes in six variants of A/Tokyo/3/67 selected with various monoclonal antibodies (Ab) to the NA were determined by dideoxy sequencing of the vRNA. In five of the monoclonal variants, a single change occurred, resulting in an amino acid substitution at residue 344. Arginine in the parent virus changed to every amino acid possible with a single nucleotide change. In another variant, arginine at position 253 changed to serine, a change that also occurred in field strains. All variants so far sequenced that were selected by monoclonal Ab to A/Tokyo/3/67 virus changed at position 344, except one which changed at residue 368. Both of these positions are in clusters of residues that vary considerably in field strains, the clusters being 344-347 and 368-370. Analysis of the three-dimensional crystal structure of the NA of A/Tokyo/3/67 shows that these clusters are directly adjacent on the protein, and likely comprise a single antigenic site. A total of three or four antigenic sites have been proposed for the NA protein, based on antigenic mapping with monoclonal Ab [R. G. Webster, V. S. Hinshaw , and W. G. Laver (1982) Virology 117, 93-104]. Variants selected by Ab to Tokyo/67 NA all change in this single antigenic site, whereas variants selected by Ab to other strains change in other regions. It is possible that, although there may be three or four antigenic sites on the NA molecule, there may be a single, dominant antigenic site for each strain.