Evolutionary dynamics of HIV-1 and the control of AIDS.

Human immunodeficiency viruses (HIV) have exhibited an extraordinary capacity for genetic change, exploring new evolutionary space after each transmission to a new host. This presents a great challenge to the prevention and management of HIV-1 infection. At the same time, the relentless diversification of HIV-1, developing as it does under the constraints imposed by the human immune system and other selective forces, contains within it information useful for understanding HIV epidemiology and pathogenesis. Comparing the sheer mutational potential of HIV with actual data representing viral lineages that can survive selection suggests that HIV does not have unlimited capacity for change. Rather, clinical and bioinformatic data suggest that, even in the most diverse gene of the most highly variable organism, natural selection places severe limits on the portion of amino acid sequence space that ensures viability. This suggests some optimism for those attempting to identify sets of antigens that can generate effective humoral and cellular immune responses against HIV.

Molecular population genetics and evolution of a prion-like protein in Saccharomyces cerevisiae.

The prion-like behavior of Sup35p, the eRF3 homolog in the yeast Saccharomyces cerevisiae, mediates the activity of the cytoplasmic nonsense suppressor known as [PSI(+)]. Sup35p is divided into three regions of distinct function. The N-terminal and middle (M) regions are required for the induction and propagation of [PSI(+)] but are not necessary for translation termination or cell viability. The C-terminal region encompasses the termination function. The existence of the N-terminal region in SUP35 homologs of other fungi has led some to suggest that this region has an adaptive function separate from translation termination. To examine this hypothesis, we sequenced portions of SUP35 in 21 strains of S. cerevisiae, including 13 clinical isolates. We analyzed nucleotide polymorphism within this species and compared it to sequence divergence from a sister species, S. paradoxus. The N domain of Sup35p is highly conserved in amino acid sequence and is highly biased in codon usage toward preferred codons. Amino acid changes are under weak purifying selection based on a quantitative analysis of polymorphism and divergence. We also conclude that the clinical strains of S. cerevisiae are not recently derived and that outcrossing between strains in S. cerevisiae may be relatively rare in nature.

Design and expression of polymeric immunoglobulin fusion proteins: a strategy for targeting low-affinity Fcgamma receptors.

We have developed a family of cloning vectors that direct expression of fusion proteins that mimic aggregated immunoglobulin (IgG) (AIG) and immune complex function with respect to their interactions with FcgammaR and that allow for the inclusion and targeting of a second protein domain to cells expressing FcgammaR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG(1) fused to the framework region of human IgG(1). Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. The resulting molecule is tripartite. The carboxyl-IgG(1) framework domain provides stability and permits dimerization, and the intervening polymer provides increased effector function and targeting to FcgammaR while the amino-terminal domain can deliver an additional signal to cells expressing FcgammaR. To demonstrate the utility of the vectors, the extracellular domain of human CD8alpha was expressed as a HCH2 polymer fusion protein. The fusion proteins were secreted in useful amounts from polyclonal cell lines established in Sf9 cells following transfection and selection with G418. The biological activity of the recombinant CD8alpha-HCH2 polymers was determined and compared to those of AIG and an anti-CD16 monoclonal antibody using an in vitro assay. The activity of the fusion proteins positively correlates to the number of HCH2 units. The largest polymer tested was severalfold more potent than AIG at similar concentrations. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcgammaR in autoimmune disorders.

Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding.

The inhibitory mechanism of serine proteinase inhibitors of the serpin family is based on their unique conformational flexibility. The formation of a stable proteinase-serpin complex implies insertion of the reactive centre loop of the serpin into the large central beta-sheet A and a shift in the relative positions of two groups of secondary structure elements, the smaller one including alpha-helix F. In order to elucidate this mechanism, we have used phage-display and alanine scanning mutagenesis to map the epitopes for four monoclonal antibodies against alpha-helix F and its flanking region in the serpin plasminogen activator inhibitor-1 (PAI-1). One of these is known to inhibit the reaction between PAI-1 and its target proteinases, an effect that is potentiated by vitronectin, a physiological carrier protein for PAI-1. When combined with the effects these antibodies have on PAI-1 activity, our epitope mapping points to the mobility of amino-acid residues in alpha-helix F and the loop connecting alpha-helix F and beta-strand 3A as being important for the inhibitory function of PAI-1. Although all antibodies reduced the affinity of PAI-1 for vitronectin, the potentiating effect of vitronectin on antibody-induced PAI-1 neutralization is based on formation of a ternary complex between antibody, PAI-1 and vitronectin, in which PAI-1 is maintained in a state behaving as a substrate for plasminogen activators. These results thus provide new details about serpin conformational changes and the regulation of PAI-1 by vitronectin and contribute to the necessary basis for rational design of drugs neutralizing PAI-1 in cancer and cardiovascular diseases.

Characterization of two-dimensional finite-aperture wire grid polarizers by a spectral-domain technique.

We investigate the transmission characteristics of perfectly conducting two-dimensional wire grid polarizers fabricated in finite and infinite apertures using a rigorous spectral-domain mode-matching method. Specifically, the transmission coefficient for both transverse-electric and transverse-magnetic polarizations, extinction ratio, and diffraction pattern are characterized for a wide variety of geometric and material parameters including aperture dimension, conducting wire fill factor, wire spacing, polarizer thickness, material dielectric constants, and incident wave arrival angle. The results indicate that the transmission behavior is largely insensitive to aperture dimension.

Finite-aperture wire grid polarizers.

The transmission characteristics of wire grid polarizers fabricated in finite apertures are investigated by using a three-dimensional finite-difference time-domain formulation. Specifically, the optical transmissivity and extinction ratio are characterized for a wide variety of geometrical parameters including aperture size in both dimensions, conducting wire fill factor, and polarizer thickness. A dispersive material model is used to investigate the performance of polarizers fabricated by using realistic metals at infrared wavelengths. The results indicate that the aperture dimension significantly impacts the polarizer transmission behavior and that the extinction of the unwanted polarization is often limited by depolarizing scattering that is due to the finite aperture size.

Influence of inorganic phosphate and energy state on force in skinned cardiac muscle from freshwater turtle and rainbow trout.

Inorganic phosphate, which increases in the hypoxic cardiac cell, depresses force development. The cardiac muscle of freshwater turtle maintains a remarkably high contractility during hypoxia; this may involve a low sensitivity to phosphate. Therefore, freshwater turtle and rainbow trout were compared with regard to Ca(2+)-activated force in skinned atrial trabeculae in a bath containing 3 mM ATP buffered by 15 mM creatine phosphate in the presence of creatine kinase. For turtle, an increase in phosphate from 0 mM to either 6 mM or 12 mM reduced maximal force by 50% and 80% respectively, whereas the Ca2+ activity eliciting half maximal force (Ca0.5) was increased by 70% in 6 mM and could not be reliably recorded in 12 mM. For trout, the effects of phosphate were less pronounced. An increase from 0 mM to 12 mM did not affect maximal force significantly, but elevated Ca0.5 by 70%. Hypoxia increases ADP as creatine phosphate is shifted to creatine, therefore, creatine phosphate was changed from 15 mM to 3 mM and creatine from 0 mM to 12 mM. After these changes, the elevation of phosphate from 0 mM to 12 mM had no significant effects for either turtle or trout. In conclusion, the high performance of turtle cardiac muscle during hypoxia does not involve a low sensitivity of the contractile system to phosphate. In addition, the effect of increased phosphate seems to be offset by a concomitant increase in ADP.

Protein interactions with the glucose transporter binding protein GLUT1CBP that provide a link between GLUT1 and the cytoskeleton.

Subcellular targeting and the activity of facilitative glucose transporters are likely to be regulated by interactions with cellular proteins. This report describes the identification and characterization of a protein, GLUT1 C-terminal binding protein (GLUT1CBP), that binds via a PDZ domain to the C terminus of GLUT1. The interaction requires the C-terminal four amino acids of GLUT1 and is isoform specific because GLUT1CBP does not interact with the C terminus of GLUT3 or GLUT4. Most rat tissues examined contain both GLUT1CBP and GLUT1 mRNA, whereas only small intestine lacked detectable GLUT1CBP protein. GLUT1CBP is also expressed in primary cultures of neurons and astrocytes, as well as in Chinese hamster ovary, 3T3-L1, Madin-Darby canine kidney, Caco-2, and pheochromocytoma-12 cell lines. GLUT1CBP is able to bind to native GLUT1 extracted from cell membranes, self-associate, or interact with the cytoskeletal proteins myosin VI, alpha-actinin-1, and the kinesin superfamily protein KIF-1B. The presence of a PDZ domain places GLUT1CBP among a growing family of structural and regulatory proteins, many of which are localized to areas of membrane specialization. This and its ability to interact with GLUT1 and cytoskeletal proteins implicate GLUT1CBP in cellular mechanisms for targeting GLUT1 to specific subcellular sites either by tethering the transporter to cytoskeletal motor proteins or by anchoring the transporter to the actin cytoskeleton.

Cytokine secretion by deltagamma and alphabeta T cells in monophasic experimental autoimmune encephalomyelitis.

Mononuclear cells were isolated from the central nervous system (CNS), lymph nodes (LN), spleen and blood, over the course of murine monophasic experimental autoimmune encephalomyelitis (EAE). Individual cytokine secreting T cells were enumerated. IL-2-secreting alphabeta T cells were numerous at all sites at disease onset. By disease peak their numbers had fallen profoundly; they remained low thereafter. IL-2 secreting gammadelta T cells were rare throughout. IFN-gamma-secreting cells were plentiful at all sites at disease onset. gammadelta T cells comprised 7% of total and 20% of IFN-gamma-secreting CNS-derived cells at disease onset; values at disease peak were 12 and 40% respectively. IL-4-secreting alphabeta T cells were rare in the CNS and LN throughout and did not increase in the spleen from baseline values. In contrast, splenic IL-4-secreting gammadelta T cells had increased to four-fold baseline values at disease onset and seven-fold at disease peak. Recovery from EAE is associated with a global inhibition of IL-2-secreting alphabeta T cells and to a lesser extent with IFN-gamma-secreting alphabeta and gammadelta T cells, whereas IL-4-secreting gammadelta T cells increase in the spleen as disease evolves.

Pronounced microsatellite instability in transitional cell carcinomas from young patients with bladder cancer.

Microsatellites may show loss of heterozygosity as well as instability of the repeats. We examined 22 different microsatellites in 14 bladder tumours (7 grade II non-invasive, 7 grade III/IV invasive) and found altered CA repeat length compared with leukocytes, indicating instability, in several microsatellites in all tumours. Instability was significantly more frequent in low stage tumours compared with high stage tumours. The number of new bands occuring was also significantly higher in low stage tumours (median 7.2) compared with high stage tumours (median 3.3). Furthermore, patients with a disease course > or = 1 year had significantly more unstable microsatellites (10.83) than those with a disease course < 1 year (mean 8.88). Examination of biopsies from normal bladder mucosa showed no instability. In 2 cases in which selected site biopsies were taken, alterations differed from the tumours, pointing at a different clonal development. LOH was most frequent in 9p markers in low stage tumours. In a group of markers located at 2p, 17p (p53), 9q, 5q and 10p, LOH was significantly more frequent in high stage tumours. Microsatellites placed at MSH2 and MLH1 loci showed LOH in several cases, indicating that the profound microsatellite instability could partly be an effect of damage to these genes.

All-trans retinoic acid potentiates the ability of interferon beta-1b to augment suppressor cell function in multiple sclerosis.

OBJECTIVE: To determine the effects of combination all-trans retinoic acid (RA) and interferon beta-1b therapy on immune system functions potentially relevant to multiple sclerosis (MS). DESIGN: Interferon gamma-secreting cells, T suppressor cell function, and lymphocyte proliferative responses were assayed using peripheral blood mononuclear cells from patients with MS and control subjects under control conditions and in the presence of interferon beta-1b, RA, and the 2 combined. SETTING: A university hospital MS clinic. PARTICIPANTS: Seventeen patients with secondarily progressive MS and 25 control subjects. RESULTS: Interferon beta-1b use increased interferon gamma-secreting cell counts, augmented T suppressor cell function, and inhibited T-cell proliferation. Therapy with RA decreased interferon gamma-secreting cell counts, had a minimal positive effect on T suppressor cell function, and had no effect on T-cell proliferation. When RA and interferon beta-1b were combined, the inhibitory effect of RA on interferon gamma-secreting cells predominated, T suppressor cell function increased synergistically over the increment observed with interferon beta-1b use alone, and the inhibitory effect of interferon beta-1b alone on T-cell proliferation remained unchanged. CONCLUSIONS: Treatment with interferon beta-1b partially restores defective T suppressor cell function in patients with MS. This potentially beneficial action is synergistically potentiated by RA. Interferon beta-1b increases the number of interferon gamma-secreting cells in the circulation when treatment is initiated. A similar increment in interferon gamma-secreting cells is observed when interferon beta-1b is added to cultural peripheral blood mononuclear cells in vitro. This potentially deleterious action of interferon beta-1b is reversed by RA. Interferon beta-1b inhibits lymphocyte proliferation modestly but reproducibly. This action of interferon beta-1b is unaltered by RA. These data provide a rationale for a trial of combination treatment with interferon beta-1b and RA in patients with MS.

Central nervous system cytokine mRNA expression following Theiler's murine encephalomyelitis virus infection.

DA strain of Theiler's murine encephalomyelitis virus (TMEV) produces a biphasic disease with an initial self-limited acute gray matter polioencephalomyelitis in all strains of mice followed by, in the case of certain susceptible strains of mice, a chronic inflammatory demyelination of the spinal cord with a persistent virus infection. A pathogenic role for T-helper 1 (Th1) cells during the demyelinating phase of disease has been proposed. We characterized the cytokine mRNA expression in the brain and spinal cord of susceptible and resistant strains of mice during the early encephalomyelitic disease and the late demyelination, using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. At the time of the encephalomyelitis, both resistant and susceptible mice expressed proinflammatory cytokine mRNAs followed by T-cell derived mRNAs; susceptible mice expressed more IL-12 p40 mRNA than resistant mice. During this early disease, there was no significant difference in Th1 cytokine mRNA expression in the brain and spinal cord among the four strains and relatively little Th2 type cytokine upregulation above levels seen in mock-infected controls. During the late demyelinating disease, susceptible but not resistant mice had evidence of viral genome and a continuous expression of Th1 type cytokine mRNAs. The expression of Th2 cytokine mRNAs varied among the different strains and did not correlate with susceptibility or resistance. The results indicate the complexity of cytokine mRNA expression following TMEV infection and the dependence of the expression on disease pathology, the time following infection and the genetics of the host.

Global inhibition of IL-2 and IFN-gamma secreting T cells precedes recovery from acute monophasic experimental autoimmune encephalomyelitis.

Actively induced experimental autoimmune encephalomyelitis (EAE) in the PL/J mouse is a monophasic disease. We isolated mononuclear cells (MNC) from the central nervous system (CNS), lymph node (LN), blood, and spleen over the course of EAE and counted the number of cells secreting IL-2, IFN-gamma or IL-4 in response to polyclonal stimulation. IL-2 secreting cells were present in the CNS at disease onset but absent at disease peak and at recovery. A profound transient drop in IL-2 secreting cells also occurred in LN, blood, and spleen at disease peak and during recovery. IFN-gamma secreting cell number decreased in all compartments as disease evolved. In contrast, IL-4 secreting cell number was greatest in the CNS at disease peak, i.e. IL-4 secreting cells rose as IL-2 and IFN-gamma secreting cells fell. IL-4 secreting cell number did not change appreciably in LN, blood, and splenic MNC as disease evolved. CNS MNC at disease peak failed to proliferate in response to anti-CD3 mAb but did so in response to IL-2. LN, blood and splenic MNC did proliferate in response to anti-CD3 mAb at disease peak and this proliferation was augmented by exogenous IL-2. After prolonged culture, proliferative response of CNS MNC to anti-CD3 mAb was restored. These results indicate that during monophasic EAE global suppression of naive T cell and Th1 T cell cytokine synthesis occurs but that T cell proliferative responsiveness is selectively inhibited in the CNS.

Use of homoduplex ribosomal DNA spacer amplification products and heteroduplex cross-hybridization products in the identification of Salmonella serovars.

When the hypervariable 16S-23S intergenic spacer regions found in prokaryotic ribosomal DNA (rDNA) are amplified from conserved adjacent sequences, homoduplex double-stranded DNA structures and heteroduplex structures containing substantial regions of single-stranded DNA are generated. The electrophoretic separation of these structures results in product profile patterns, which may be organized into highly correlated pattern groups of ribosomal spacer and heteroduplex polymorphism (RS/HP) types. In a test panel of 380 Salmonella strains that were analyzed by this procedure, 36 unique RS/HP types were observed. Of the 28 serovars in the test group, 21 showed single characteristic RS/HP types. The remaining seven serovars each contained multiple RS/HP types, which were also unique to individual serovars. Formation of heteroduplex structures with a substantially reduced electrophoretic mobility was observed in 29 of the 36 RS/HP pattern types. Because the mobility of these heteroduplex structures is sensitive to intergenic spacer sequence composition, the presence of these structures adds an additional diagnostic feature that is extremely useful in the differentiation of Salmonella serovars. The RS/HP types show sufficient diversity to be useful in the identification of many commonly observed Salmonella serovars. This analytical procedure is simple to perform and is well suited to rapid and inexpensive screening of large numbers of Salmonella strains.

Interferon-gamma-secreting cells in multiple sclerosis patients treated with interferon beta-1b.

Fifteen percent of multiple sclerosis patients about to be treated with interferon beta-1b exhibited elevated numbers of circulating interferon-gamma-secreting cells, defined as a value that exceeded the mean value for healthy controls by more than two standard deviations. Sixty percent of patients receiving the drug exhibited elevated interferon-gamma-secreting cell numbers during their first 2 months of treatment. Values normalized after 3 months. Prednisone treatment during the first month on the drug prevented the interferon-gamma-secreting cell surge.

A reduction in serum glucocorticoids provokes experimental allergic encephalomyelitis: implications for treatment of inflammatory brain disease.

Glucocorticoid (GCC) therapy usually inhibits inflammatory diseases, but certain regimens can trigger relapses. Clinical use of steroids is not uniform and in some instances may be dangerous. In the present study, GCCs modified the course of experimental allergic encephalomyelitis (EAE) in Lewis rats, a model of inflammatory CNS disease. Continuous treatment with dexamethasone (DEX) completely blocked EAE. RU 486, a GCC antagonist, counteracted the effects of endogenous GCCs and worsened EAE. Sudden withdrawal of DEX also caused severe clinical and histologic exacerbations at a time when paired saline-treated animals had completely recovered. In rats that had complete clinical recovery from EAE, and would not have relapsed without this acute steroid deficit, a short pulse of DEX was followed by severe exacerbations. In contrast, a slow steroid taper prevented exacerbations. Abrupt discontinuation of GCCs provokes inflammatory brain disease.

Prostaglandins and inhibitors of arachidonate metabolism suppress experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS). It is an animal model of post-infectious encephalomyelitis and multiple sclerosis (MS). Acute EAE is mediated by macrophages and by T helper 1 (Th1) lymphocytes directed against brain antigens. Inflammation in EAE could potentially be modified by prostaglandins (PG) secreted by blood monocytes (Mo) and brain glial cells. PGE elevates cAMP, which inhibits Mo function and selectively blocks secretion of cytokines by Th1 cells. In the present study, we found that a long-acting PGE1 analogue (LAPGE) inhibited clinical and histological EAE. Indomethacin (INDO) also suppressed active EAE. The combination of INDO plus LAPGE inhibited disease further, possibly by allowing LAPGE to function unopposed by immunostimulatory PG. EAE was suppressed when these agents were administered from the time of immunization or from the onset of clinical disease. The combination of INDO plus LAPGE also inhibited delayed-type hypersensitivity (DTH) reactions to myelin basic protein (MBP), and diminished in vitro lymphocyte responses to mitogens and MBP. PGE analogues and modifiers of arachidonate metabolism block autoimmune responses to brain antigens in vitro and in vivo, and may ameliorate inflammatory and autoimmune diseases of the brain and other organs.

Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions.

PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Single-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a single-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer/template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions.

Astrocyte cytolysis by MHC class II-specific mouse T cell clones.

The brain is "immunologically privileged," in part because class I and II MHC antigens are not normally present on glia or neurons. However, under certain conditions such as transplantation, glial cells express MHC proteins at levels sufficient for glia to become targets of immune responses. Cultured astrocytes expressing very low levels of MHC class I protein are killed efficiently by MHC class I antigen-specific CTL. Mouse brain allografts, however, are rejected by CD4+ T cells that are likely to be class II MHC-specific. The level of expression of MHC class II antigen needed to trigger specific killing of astrocytes by CD4+ T cells, independent of exogenous antigen, has not been studied. We examined the role of glial class II MHC in the lysis of cultured neonatal mouse astrocytes by an alloreactive murine CD4+ CTL alone. IFN-gamma induced functionally relevant increases in MHC class II antigen on target cells. Astrocytes were lysed by the CD4+ clone only when class II MHC antigens reached levels readily detectable by flow cytometry. MHC class II expression and lysis increased when astrocytes were coincubated with IFN-gamma and TNF-alpha. Conversely, lysis decreased when class II expression was downregulated by IFN-alpha/beta or dbcAMP. Cytolysis by CD4+ clones was blocked by antibodies to CD4 and LFA-1 on T cells, and to ICAM-1 and class II molecules on astrocytes. The role of LFA-1 in CD4+ cell-mediated lysis was greater than that of LFA-1/ICAM-1 in CD8+ T cell-mediated lysis. CD4+ cells may lyse activated astrocytes when the immune privilege of the brain is compromised as in transplantation, tumors, and inflammatory diseases.