Two orphan seven-transmembrane segment receptors which are expressed in CD4-positive cells support simian immunodeficiency virus infection.

Clinical isolates of primate immunodeficiency viruses, including human immunodeficiency virus type 1 (HIV-1), enter target cells by sequential binding to CD4 and the chemokine receptor CCR5, a member of the seven-transmembrane receptor family. HIV-1 variants which use additional chemokine receptors are present in the central nervous system or emerge during the course of infection. Simian immunodeficiency viruses (SIV) have been shown to use CCR5 as a coreceptor, but no other receptors for these viruses have been identified. Here we show that two orphan seven-transmembrane segment receptors, gpr1 and gpr15, serve as coreceptors for SIV, and are expressed in human alveolar macrophages. The more efficient of these, gpr15, is also expressed in human CD4(+) T lymphocytes and activated rhesus macaque peripheral blood mononuclear cells. The gpr15 and gpr1 proteins lack several hallmarks of chemokine receptors, but share with CCR5 an amino-terminal motif rich in tyrosine residues. These results underscore the potential diversity of seven-transmembrane segment receptors used as entry cofactors by primate immunodeficiency viruses, and may contribute to an understanding of viral variation and pathogenesis.

The envelope glycoprotein ectodomains determine the efficiency of CD4+ T lymphocyte depletion in simian-human immunodeficiency virus-infected macaques.

CD4+ T lymphocyte depletion in human immunodeficiency virus type 1 (HIV-1)-infected humans underlies the development of acquired immune deficiency syndrome. Using a model in which rhesus macaques were infected with chimeric simian-human immunodeficiency viruses (SHIVs), we show that both the level of viremia and the structure of the HIV-1 envelope glycoprotein ectodomains individually contributed to the efficiency with which CD4(+) T lymphocytes were depleted. The envelope glycoproteins of recombinant SHIVs that efficiently caused loss of CD4(+) T lymphocytes exhibited increased chemokine receptor binding and membrane-fusing capacity compared with those of less pathogenic viruses. These studies identify the HIV-1 envelope glycoprotein ectodomains as determinants of CD4(+) T lymphocyte loss in vivo and provide a foundation for studying pathogenic mechanisms.

CD4-independent binding of SIV gp120 to rhesus CCR5.

CCR5 and CD4 are coreceptors for immunodeficiency virus entry into target cells. The gp120 envelope glycoprotein from human immunodeficiency virus strain HIV-1(YU2) bound human CCR5 (CCR5hu) or rhesus macaque CCR5 (CCR5rh) only in the presence of CD4. The gp120 from simian immunodeficiency virus strain SIVmac239 bound CCR5rh without CD4, but CCR5hu remained CD4-dependent. The CD4-independent binding of SIVmac239 gp120 depended on a single amino acid, Asp13, in the CCR5rh amino-terminus. Thus, CCR5-binding moieties on the immunodeficiency virus envelope glycoprotein can be generated by interaction with CD4 or by direct interaction with the CCR5 amino-terminus. These results may have implications for the evolution of receptor use among lentiviruses as well as utility in the development of effective intervention.

Maternal polymorphisms in the FADS1 and FADS2 genes modify the association between PUFA ingestion and plasma concentrations of omega-3 polyunsaturated fatty acids.

There is a lack of studies about polymorphisms in FADS genes in pregnant women. We aimed to verify the interaction between three FADS gene polymorphisms (rs174561; rs174575; rs3834458) and dietary α-linolenic acid (ALA) or linoleic/α-linolenic acid ratio (LA/ALA) and plasma concentrations of omega-3 (n-3) PUFAs in pregnant women. Of the 250 women evaluated, the homozygous for the rs174561 and rs3834458 minor allele had high plasma ALA concentrations at the highest ALA and LA/ALA ratio tertile (p < 0.05). Plasma concentrations of EPA and DHA were not influenced by diet. For the rs174575 SNP, pregnant women who carried the minor allele presented lower proportions of plasma EPA in the second LA/ALA ratio tertile (p < 0.05). Increased dietary intake of ALA and LA/ALA ratio promoted plasma ALA accumulation in homozygotes for the minor allele rs174561 and rs3834458. Moderate intake of LA/ALA ratio may reduce plasma concentration of EPA in pregnants carrying the rs174575 minor allele.

Optimization of a single chamber microbial fuel cell using Lactobacillus pentosus: Influence of design and operating parameters.

Microbial fuel cells (MFCs) have been receiving an increased attention over the last years due to their potential to combat two global problems: waste pollution and energy demand. Additionally, when a wastewater is used, MFCs can perform its treatment while recovering energy, leading to the possibility of energy-producing wastewater treatment plants, offsetting their operational costs. However, to overcome their current limitations (lower power outputs and higher costs), a clear understanding of the effect of operation and design parameters on its overall performance is mandatory. Therefore, the goal of this work was to evaluate the effect of operating conditions - batch cycle and yeast extract concentration, and design parameters - anode electrode area, membrane thickness and active area, on the overall performance of a single chamber MFC. The MFC operated with a pure culture of Lactobacillus pentosus and a synthetic wastewater based on a real dairy industry effluent. The overall performance was evaluated through the power output and the COD removal rate. Additionally, the biofilm formed at the anode electrode was characterized in terms of biomass, proteins and polysaccharides content. For the conditions used in this work, a maximum power density of 5.04 ±â€¯0.39 mW/m2 was achieved with an anode electrode area of 61 cm2, a batch cycle of 48 h, 50 mg/L of yeast extract and a Nafion 212 membrane with an active area of 25 cm2. The different conditions tested had a clear effect on the MFC energy production and biofilm characteristics, but not on the ability of L. pentosus to treat the dairy wastewater. The COD removal rates were in the range between 42% and 58%, for all the conditions tested.

Self-crosslinked fibrous collagen/chitosan blends: Processing, properties evaluation and monitoring of degradation by bi-fluorescence imaging.

Porous collagen/chitosan scaffolds with different Collagen:Chitosan (Coll:Ch) ratios were prepared by freeze-drying followed by self-crosslinking via dehydrothermal treatment (DHT) and characterized as biomaterials for tissue engineering. Cy7 and Cy5.5 fluorochromes were covalently grafted to collagen and chitosan, respectively. Thus, it was possible, using optical fluorescence imaging of the two fluorochromes, to simultaneously track their in vivo biodegradation, in a blend scaffold form. The fluorescence signal evolution, due to the bioresorption, corroborated with histological analysis. In vitro cytocompatibility of Coll:Ch blend scaffolds were evaluated with standardized tests. In addition, the scaffolds showed a highly interconnected porous structure. Extent of crosslinking was analyzed by convergent analysis using thermogravimetry, Fourier Transform Infrared Spectroscopy and PBS uptake. The variations observed with these techniques indicate strong interactions between collagen and chitosan (covalent and hydrogen bonds) promoted by the DHT. The mechanical properties were characterized to elucidate the impact of the different processing steps in the sample preparation (DHT, neutralization and sterilization by ß-irradiation) and showed a robust processing scheme with low impact of Coll:Ch composition ratio.

Immunomodulatory properties and toxicity of interleukin 2 in patients with cancer.

We performed a phase Ia/Ib study of interleukin 2 (IL2) in patients with cancer. Single doses of IL2 from 10(3) units/m2 to 10(7) units/m2 were well tolerated but failed to induce significant immunological changes. Chronic IL2 treatment for 5 days out of 7 for 3 weeks was well tolerated at doses below 10(7) units/m2 and was accompanied by significant immunological changes. Following chronic treatment with intramuscular injections of IL2 at 1 x 10(6) units/m2, we observed augmentation of peripheral blood natural killer activity and induction of peripheral blood LAK activity. Induction of LAK activity was most evident when IL2 was included in the cytotoxicity assay. There was a marked increase in the number of peripheral blood mononuclear cells bearing the Leu-19 marker in association with the observed increases in natural killer and LAK activity. A small percentage of Leu-19+ cells coexpressed CD3. There was heterogeneous expression of the low affinity Fc receptor (CD16). In vivo induced Leu-19+ cells could be divided into two populations, dim and bright, based on the intensity of fluorescent staining with antibodies to Leu-19. The majority of Leu-19 bright cells were CD16- while the majority of Leu-19 dim cells were CD16+. In addition, the intensity of CD16 staining was higher for Leu-19 dim cells than for Leu-19 bright cells. Increases in the amounts of CD38 and CD8 antigens were also observed. Significant increases in serum levels of the soluble IL2 receptor were observed during treatment. One partial remission was noted in a woman with non-Hodgkin's lymphoma.

A competitive enzyme-linked immunoassay (ELISA) for the measurement of soluble human interleukin-2 receptors (IL-2R, Tac protein).

A solid-phase, competition enzyme-linked immunosorbent assay (ELISA) was established for the quantitative measurement of soluble (human) interleukin-2 receptors (IL-2R). The ladder of reagents from the solid phase up consisted of: (1) recombinant DNA-derived, purified IL-2R, (2) sample-containing soluble IL-2R and fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody, 7G7/B6, directed against the IL-2R, (3) alkaline phosphatase-conjugated rabbit anti-FITC, and (4) substrate. This ELISA was compared with a 'sandwich' ELISA for soluble IL-2R. The competitive ELISA was less sensitive than the 'sandwich' assay, being capable of measuring 5000 versus 31 U/ml, respectively. While both anti-Tac and 7G7/B6 in the IL-2R-containing sample inhibited the 'sandwich' assay, only 7G7/B6 inhibited the competition assay. Anti-mouse immunoglobulin enhanced the 'sandwich' assay and inhibited the competitive assay; both effects could be overcome by the addition of normal mouse immunoglobulin in the sample buffer. Studies of a patient's serum receiving anti-Tac as therapy for the adult T cell leukemia demonstrated that rises in the level of IL-2R occurring with anti-Tac therapy, as measured with the competition assay, were masked in the 'sandwich' assay. This competition ELISA will be useful for measuring soluble IL-2R levels in patients receiving anti-Tac as therapy for various immunologic disorders.

Projections from the reticular formation of the medulla, the spinal trigeminal and lateral reticular nuclei to the inferior olive.

Injections of tritiated L-leucine were placed in the reticular formation of the medulla, the spinal trigeminal and lateral reticular nuclei of cats and silver grain accumulations in the inferior olivary nucleus were demonstrated by autoradiography. Cells of the reticular formation located at the junction of nuclei reticularis magnocellularis and reticularis parvocellularis in the rostral medulla and within nucleus reticularis ventralis in the caudal medulla contribute four distinct projections to the olive. Three projections are distributed ipsilaterally in the caudal part of the medial accessory olive, at mid-level of the dorsal accessory olive and in the ventrolateral bend of the principal olive, at rostral levels. There is also a small controlateral projection to the caudal part of the medial accessory olive. the spinal trigeminal nucleus sends crossed projections to the rostral part of the dorsal accessory olive and adjacent ventral lamella as well as to the caudal part of the medial accessory olive. The lateral reticular nucleus sends an extensive ipsilateral projection to the caudal part of the medial accessory olive and provides a small contribution to the same subdivision, contralaterally. All these projections converge with other known afferents to the olive.

High degree of sensitivity of the simian immunodeficiency virus (SIVmac) envelope glycoprotein subunit association to amino acid changes in the glycoprotein 41 ectodomain.

The infection of macaques by simian immunodeficiency virus (SIVmac) represents an attractive model to study the pathogenic determinants of primate and human immunodeficiency viruses. The utility of this model would be enhanced if genetic changes in human immunodeficiency virus (HIV-1) associated with interesting in vitro properties would, when introduced into SIVmac, result in similar phenotypes. In this study, we introduced amino acid changes into the SIVmac239 envelope glycoproteins that, in the context of the HIV-1 envelope glycoproteins, disproportionately attenuated in vitro cytopathic effects compared with the viral replication rate. Amino acid changes in the SIVmac239 gp41 ectodomain altered the noncovalent association of the gp120 and gp41 glycoproteins significantly more than did analogous changes in the HIV-1 envelope glycoproteins. Decreases in the affinity of the gp120-gp41 interaction were observed and were associated with a dramatic attenuation of virus replication not seen in the HIV-1 studies. The increased sensitivity of the SIVmac gp120-gp41 interaction to amino acid changes presents an obstacle to the direct extension of results obtained with the HIV-1 envelope glycoproteins to the SIVmacaque model.

Modeling digits. Digit patterning is controlled by a Bmp-Sox9-Wnt Turing network modulated by morphogen gradients.

During limb development, digits emerge from the undifferentiated mesenchymal tissue that constitutes the limb bud. It has been proposed that this process is controlled by a self-organizing Turing mechanism, whereby diffusible molecules interact to produce a periodic pattern of digital and interdigital fates. However, the identities of the molecules remain unknown. By combining experiments and modeling, we reveal evidence that a Turing network implemented by Bmp, Sox9, and Wnt drives digit specification. We develop a realistic two-dimensional simulation of digit patterning and show that this network, when modulated by morphogen gradients, recapitulates the expression patterns of Sox9 in the wild type and in perturbation experiments. Our systems biology approach reveals how a combination of growth, morphogen gradients, and a self-organizing Turing network can achieve robust and reproducible pattern formation.

Fluidics of a nanogap.

We have determined the filling properties of nanogaps with chemically heterogeneous walls. The quantitative criteria we present allow the prediction of the liquid loading of the nanostructure. They can easily be applied in combination with contact-angle measurements on planar substrates of the nanogap materials. We present an application of the theory to a recently developed nanogap biosensor. Chemical force microscopy (CFM) is employed to characterize the initial silanol properties of the gap. The functionality of the complex surface chemistry of the biosensor is demonstrated by the observation of functionalized nanoparticles in the gap with its resulting characteristic current-voltage relationship.

gp120-independent fusion mediated by the human immunodeficiency virus type 1 gp41 envelope glycoprotein: a reassessment.

In a natural context, membrane fusion mediated by the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins involves both the exterior envelope glycoprotein (gp120) and the transmembrane glycoprotein (gp41). Perez et al. (J. Virol. 66:4134-4143, 1992) reported that a mutant HIV-1 envelope glycoprotein containing only the signal peptide and carboxyl terminus of the gp120 exterior glycoprotein fused to the complete gp41 glycoprotein was properly cleaved and that the resultant gp41 glycoprotein was able to induce the fusion of even CD4-negative cells. In the studies reported herein, mutant proteins identical or similar to those studied by Perez et al. lacked detectable cell fusion activity. The proteolytic processing of these proteins was very inefficient, and one processed product identified by Perez et al. as the authentic gp41 glycoprotein was shown to contain carboxyl-terminal gp120 sequences. Furthermore, no fusion activity was observed for gp41 glycoproteins exposed after shedding of the gp120 glycoprotein by soluble CD4. Thus, evidence supporting a gp120-independent cell fusion activity for the HIV-1 gp41 glycoprotein is currently lacking.

Dispensable role of the human immunodeficiency virus type 2 Vpx protein in viral replication.

Human immunodeficiency virus type 2 (HIV-2) is similar in genetic organization to HIV-1 but contains a unique gene (vpx) that encodes a 16-kDa protein. A replication-competent molecular clone of HIV-2 (HIV-2sbl/isy) that infects human primary cells in vitro and rhesus monkeys was used to generate three mutations in the vpx gene. In the first mutant, the vpx open reading frame was truncated at amino acid 20; the second mutant was tailored to eliminate the proline-rich carboxyl terminus of the protein; and the third mutant was obtained by addition of four amino acids (KDEL) to the carboxyl terminus of the protein to provide a retention signal in the endoplasmic reticulum. The viral infection kinetics of the three mutant viruses and isogeneic HIV-2sbl/isy in the SupT1 cell line were similar. Slight impairment in the early phases of viral replication was observed during infection of primary human peripheral blood mononuclear cells with the vpx mutant viruses. All of the vpx mutant viruses readily infected macrophages, indicating that vpx expression is dispensable for HIV-2 infection and replication in human macrophages.

Soluble Tac peptide is present in the urine of normal individuals and at elevated levels in patients with adult T cell leukaemia (ATL).

The T lymphocyte-derived lymphokine interleukin 2 and the cell-associated receptor for this molecule play major roles in the activation and regulation of the human immune response. An enzyme-linked immunosorbent assay has been developed to measure quantitatively a soluble form of one component of the human interleukin 2 receptor, namely the Tac peptide. In the present studies, soluble Tac peptide was measured in the urine of normal individuals (mean = 92 U/ml), a level not significantly different (0.01 less than P less than 0.05) from the corresponding serum concentrations (mean = 175). The urinary Tac peptide had a molecular weight of 40-45 kD by sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis and specifically bound interleukin 2. Elevated levels of urinary Tac peptide were found in four patients with adult T cell leukaemia who also had elevated serum levels of Tac peptide. Thus, urine may represent a valuable source of lymphokine-binding proteins that may serve as important markers of immunological activation.

The human immunodeficiency virus type 2 vpr gene is essential for productive infection of human macrophages.

The human immunodeficiency virus (HIV) genetic determinant(s) responsible for tropism in human T cells or macrophages are not well defined. We studied the role of the HIV type 2 (HIV-2) nef and vpr genes in viral tropism. HIV-2 mutants, lacking either vpr or nef genes, or both vpr and nef, were obtained by site-specific mutagenesis of a biologically active HIV-2 proviral clone (HIV-2sbl/isy), which is infectious in both human T cells and macrophages. Viral progeny carrying mutations of nef, vpr, or of both nef and vpr genes replicated more efficiently than the parental virus in primary human peripheral blood cells and in the human Hut 78 T-cell line. In contrast, the HIV-2 nef- mutant infected human macrophages as efficiently as the parental virus, whereas viruses lacking the vpr gene either alone or in conjunction with the lack of the nef gene did not replicate in macrophages. Thus, some lack of nef in HIV-2 enhances viral replication in T cells and does not interfere with viral replication in primary macrophages, whereas vpr is essential for replication of HIV-2 in human macrophages. Because the parental HIV-2sbl/isy cloned virus also infects rhesus macaques, the use in animal studies of these HIV-2 mutants with differences in cell tropism and rates of replication will be highly useful in understanding the mechanism of viral infectivity and possibly pathogenicity in vivo.

Effects of soluble CD4 on simian immunodeficiency virus infection of CD4-positive and CD4-negative cells.

A soluble form of the CD4 receptor (sCD4) can either enhance or inhibit the infection of cells by simian immunodeficiency virus (SIV) and human immunodeficiency virus. We investigated the basis for these varying effects by studying the entry of three SIV isolates into CD4-positive and CD4-negative cells expressing different chemokine receptors. Infection of CD4-negative cells depended upon the viral envelope glycoproteins and upon the chemokine receptor, with CCR5 and gpr15 being more efficient than STRL33. Likewise, enhancement of infection by sCD4 was observed when CCR5- and gpr15-expressing target cells were used but not when those expressing STRL33 were used. The sCD4-mediated enhancement of virus infection of CD4-negative, CCR5-positive cells was related to the sCD4-induced increase in binding of the viral gp120 envelope glycoprotein to CCR5. Inhibitory effects of sCD4 could largely be explained by competition for virus attachment to cellular CD4 rather than other detrimental effects on virus infectivity (e.g., disruption of the envelope glycoprotein spike). Consistent with this, the sCD4-activated SIV envelope glycoprotein intermediate on the virus was long-lived. Thus, the net effect of sCD4 on SIV infectivity appears to depend upon the degree of enhancement of chemokine receptor binding and upon the efficiency of competition for cellular CD4.

Elevated concentrations of soluble interleukin-2 receptors in serum samples and bronchoalveolar lavage fluids in active sarcoidosis.

Sarcoidosis is a granulomatous disorder of unknown cause characterized by activation of T-lymphocytes. We here report the use of an enzyme-linked immunosorbent assay for the soluble interleukin-2 receptor (IL-2R) as a measure of T-cell activation in serum samples and bronchoalveolar lavage fluids in 15 patients with active sarcoidosis. The geometric mean (x divided by SEM) value for soluble IL-2R in serum samples from patients with sarcoidosis was 1,110 (x divided by 1.17) versus 224 (x divided by 1.08) U/ml for normal control subjects (p less than 0.001). Detectable levels of soluble IL-2R were present in bronchoalveolar lavage fluids from 10 of 15 patients with sarcoidosis versus only 2 of 36 normal control subjects (p less than 0.001). Levels of soluble IL-2R in serum samples from untreated patients with sarcoidosis correlated with 67gallium lung scanning scores (p less than 0.05) but not with serum angiotensin-converting enzyme concentrations or constituents of bronchoalveolar lavage. In 5 patients, the level of soluble IL-2R in serum samples fell from 1,499 (x divided by 1.20) to 476 (x divided by 1.58) U/ml (p less than 0.05) after 6 wk of successful treatment with corticosteroids, whereas the changes in soluble IL-2R in bronchoalveolar lavage fluids were more variable. These observations suggest that measurements of soluble IL-2R, particularly in serum samples, may reflect disease activity and be clinically useful in the management of patients with sarcoidosis.