Nitrogen acquisition and resource allocation strategies in temperate seagrass Zostera nigricaulis: Uptake, assimilation and translocation processes.

The dominant seagrass in Port Phillip Bay (PPB), Australia, Zostera nigricaulis, declined between 2000 and 2011, coinciding with the 'Millennium drought' that ended in 2009. These seagrasses are nitrogen-limited, underpinning the need to develop nitrogen budgets for better ecosystem management. Environmentally realistic measurements of specific uptake rates and resource allocation were undertaken to develop nitrogen budgets and test the hypothesis that the above-ground and below-ground compartments are able to re-mobilise ammonium and nitrate through uptake, translocation and assimilation to adapt to varying levels of nitrogen in the ecosystem. Uptake of 15N labelled ammonium and nitrate by above- and below-ground seagrass biomass, epiphytes and phytoplankton was quantified in chambers in situ. Preferential uptake of ammonium over nitrate was observed, where the uptake rate for nitrate was about one sixth of that for ammonium. Epiphytes and phytoplankton also registered an increased affinity for ammonium over nitrate. Translocation experiments demonstrated the uptake by both the above-ground and below-ground biomass, respectively from the water column and pore water, and subsequent translocation to the opposite compartment. Acropetal translocation (below- to above-ground biomass) was more prevalent than basipetal translocation. This is a unique outcome given basipetal translocation has been widely reported for Zostera by other researchers.

Inhibition of IκB kinase by thalidomide increases hepatitis C virus RNA replication.

Hepatic fibrosis is an integral element in the progression of chronic liver disease. Elevated hepatic interleukin (IL)-8 is an important contributor to fibrosis in patients chronically infected with the hepatitis C virus (HCV). Thalidomide has been used to reduce liver inflammation and fibrosis in HCV-infected patients, but its impact on HCV replication remains unclear. This study examined the effect of thalidomide on HCV replication in vitro. Results revealed that while thalidomide reduced IL-8 and nuclear factor kappa B (NF-κB) activity by 95% and 46% in Huh-7 cells, increasing concentrations of thalidomide correlated with a linear rise in HCV replication (17-fold at 200 µm). The NF-κB inhibitors, wedelolactone and NF-κB activation inhibitor-1, which mimic the actions of thalidomide by preventing phosphorylation and activation of IκB kinase (IKK) and hence block NF-κB activity, increased HCV RNA by 18- and 19-fold, respectively. During in vitro HCV replication in Huh-7 cells, we observed a 30% increase in IKKα protein and 55% decrease in NF-κB(p65)/RelA protein relative to cellular ß-actin. Ectopic expression of IKKα to enhance the inactive form of IKK in cells undergoing virus replication led to a 13-fold increase in HCV RNA. Conversely, enhanced expression of NF-κB(p65)/RelA in infected cells resulted in a 17-fold reduction in HCV RNA. In conclusion, HCV RNA replication was significantly augmented by the inhibition of IKK activation and subsequent NF-κB signalling, whereas a restoration of NF-κB activity by the addition of NF-κB/RelA markedly reduced HCV replication. This study lends added importance to the role of the NF-κB signalling pathway in controlling HCV replication.

Gestural imitation by a Gorilla: evidence and nature of the capacity / Imitación gestual por un gorila: evidencia y naturaleza de la capacidad

We report the results of an opportunistic experiment on the capacity for gestural imitation in a zoo-housed, female western low land gorilla (Gorilla g. gorilla). Taking advantage of her temporary disposition to copy humans, we presented 7 non-species-typical gestures, without training or rewards. The gorilla’s behaviour was filmed and subsequently rated for gestural imitation by 20 naïve coders, controlling for general demeanour by comparing pre- and post-demonstration segments. For several gestures, behaviour that closely matched the demonstration was seen only or more often after demonstration: gestural imitation was therefore reliably detected. Nevertheless, as in previous studies of great ape gestura limitation, none of the gorilla copies was a perfect match. We suggest that gestural imitation in great apes is based on facilitation of rare behaviours in their extensive and often idiosyncratic gestural repertoire (e.g. by mirror neurons), rather than on acquiring novel behaviours by imitation

Presentamos los resultados de un experimento oportunista sobre la capacidad para la imitación de gestos de una gorila de costa (Gorilla gorilla gorilla) alojada en un zoo. Aprovechando su disposición temporal a copiar a seres humanos, presentamos 7 gestos no específicos, sin entrenamiento ni recompensas. El comportamiento de la gorila fue filmado y puntuado en relación a la capacidad de imitación gestual por 20 codificadores que carecían de experiencia previa en observación de conducta animal y/o gorilas. Para ello, compararon los segmentos pre y post-demostración del modelo humano a imitar. Para varios gestos, la conducta que igualaba muy de cerca a la demostración, fue observada sólo o más amenudo después de presentada la demostración: la imitación gestual por lo tanto fue detectada de forma confiable. Sin embargo, como en estudios anteriores de la imitación de gestos en los grandes simios, ningunas de las imitaciones de la gorila fueron una copia perfecta. Sugerimos que la imitación de gestos por los grandes simios está basada más en la facilitación de comportamientos raros de su amplio y a menudo idiosincrásico repertorio de gestos (e.g., por las neuronas espejo) más que en la adquisición de nuevas conductas por imitación

Cleavage of intracellular hepatitis C RNA in the virus core protein coding region by deoxyribozymes.

Hepatitis C virus (HCV) infection represents an important global health problem. Current antiviral therapeutics for HCV have proven inadequate in stemming the disease process. A novel therapeutic strategy involves the use of deoxyribozymes, also known as DNA enzymes or DNAzymes. These catalytic DNA molecules, designed to target and cleave specific RNA sequences, have shown promise in in vitro experimental models for various diseases and may serve as an alternative or adjunct to current HCV drug therapy. We designed and tested several deoxyribozymes that can bind and cleave highly conserved RNA sequences encoding the HCV core protein in in vitro systems. One of these deoxyribozymes reduced the level of our HCV RNA target by 32% and 48% after 24 h of cell exposure when tested in human hepatoma and epithelial cell lines, respectively. As this deoxyribozyme showed significant cleavage activity against HCV core protein target RNA in human cells, it may have potential as a therapeutic candidate for clinical trial in HCV infected patients.

Mutational analysis of P-glycoprotein: suppression of caspase activation in the absence of ATP-dependent drug efflux.

P-glycoprotein (P-gp) can induce multidrug resistance (MDR) through the ATP-dependent efflux of chemotherapeutic agents. We have previously shown that P-gp can inhibit nondrug apoptotic stimuli by suppressing the activation of caspases. To determine if this additional activity is functionally linked to ATP hydrolysis, we expressed wild-type and ATPase-mutant P-gp and showed that cells expressing mutant P-gp could not efflux chemotherapeutic drugs but remained relatively resistant to apoptosis. CEM lymphoma cells expressing mutant P-gp treated with vincristine showed a decrease in the fraction of cells with apoptotic morphology, cytochrome c release from the mitochondria and suppression of caspase activation, yet still accumulated in mitosis and showed a loss of clonogenic potential. The loss of clonogenicity in vincristine-treated cells expressing mutant P-gp was associated with accumulation of cells in mitosis and the presence of multinucleated cells consistent with mitotic catastrophe. The antiapoptotic effect of mutant P-gp was not affected by antibodies that inhibit the efflux function of the protein. These data are consistent with a dual activity model for P-gp-induced MDR involving both ATPase-dependent drug efflux and ATPase-independent inhibition of apoptosis. The structure-function analyses described herein provide novel insight into the mechanisms of action of P-gp in mediating MDR.

Active habitat selection for sand by juvenile western king prawns, Melicertus latisulcatus (Kishinouye).

This paper presents the results of a series of habitat selection experiments aimed at determining if juvenile Melicertus latisulcatus generally occur on intertidal sand- and mud-flats as a result of active selection of unvegetated areas, or due to extrinsic factors (e.g. differential predation). In the laboratory, juvenile M. latisulcatus showed a clear preference for habitats containing sand irrespective of the presence or absence of predators. If sand was not available, artificial seagrass was chosen as a secondary preference but was avoided when sand alone was also present. Importantly, the combinations of habitats chosen for testing allowed us to determine that artificial seagrass provided a good surrogate for real seagrass, and that the presence of potential food (epiphytes) did not appear to influence habitat selection. There was also no difference in the habitat selected between day and night, and only minor differences with prawn size. Thus, juvenile M. latisulcatus appear to have a hierarchy of mechanisms for avoiding predators, with burying in sand being the preferred option. If burying is not possible, then seagrass is used for shelter. Active habitat selection to avoid predation appears likely to play a substantial role in determining the distribution of these animals on unvegetated sand- and mud-flats.

The Epstein-Barr virus and post-transplant lymphoproliferative disease: interplay of immunosuppression, EBV, and the immune system in disease pathogenesis.

Transplant patients are at particular risk for developing post-transplant lymphoproliferative disease (PTLD) following administration of immunosuppressive therapy. In many cases the PTLD lesions express Epstein-Barr virus (EBV) latent and lytic genes as well as elevated levels of host cytokines. An outline of the potential contributions of EBV, host cytokines and T cells, and the immunosuppressive cyclosporine A, tacrolimus, and anti-CD3 antibody in the mechanism and pathogenesis of this disease is presented and discussed.

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.

Activation of transcription factors NF-kappaB and NF-IL-6 by human immunodeficiency virus type 1 protein R (Vpr) induces interleukin-8 expression.

Human immunodeficiency virus (HIV)-positive individuals express elevated levels of interleukin-8 (IL-8), which is believed to be responsible for some of the clinical manifestations occurring during AIDS. We report here that virion-derived HIV type 1 (HIV-1) protein R (Vpr) increased IL-8 expression in primary T cells and macrophages, as well as in the T-cell line Jurkat, the monocytic cell line U937, and the epithelial cell line A549. Vpr appeared to increase IL-8 expression and IL-8 promoter activity by activating transcription factors NF-kappaB and NF-IL-6. Elevated Vpr was also shown to increase transcription of the NF-kappaB and NF-IL-6 enhancer-containing viral promoters for HIV, cytomegalovirus, and simian virus 40, as well as increase the expression of IL-6 and IL-10 in primary macrophages and in A549 cells, tumor necrosis factor alpha expression in primary T cells, and IL-6 and gamma interferon expression in U937 cells. These results suggest a new role for Vpr in the pathogenesis of HIV infection, namely, the activation of transcription factors NF-IL-6 and NF-kappaB.

International Arctic Seas Assessment Project.

The International Atomic Energy Agency responded to the news that the former Soviet Union had dumped radioactive wastes in the shallow waters of the Arctic Seas, by launching the International Arctic Seas Assessment Project in 1993. The project had two objectives: to assess the risks to human health and to the environment associated with the radioactive wastes dumped in the Kara and Barents Seas; and to examine possible remedial actions related to the dumped wastes and to advise on whether they are necessary and justified. The current radiological situation in the Arctic waters was examined to assess whether there is any evidence for releases from the dumped waste. Potential future releases from the dumped wastes were predicted, concentrating on the high-level waste objects containing the major part of the radionuclide inventory of the wastes. Environmental transport of released radionuclides was modelled and the associated radiological impact on humans and the biota was assessed. The feasibility, costs and benefits of possible remedial measures applied to a selected high-level waste object were examined. Releases from identified dumped objects were found to be small and localised to the immediate vicinity of the dumping sites. Projected future annual doses to members of the public in typical local population groups were very small, less than 1 microSv--corresponding to a trivial risk. Projected future doses to a hypothetical group of military personnel patrolling the foreshore of the fjords in which wastes have been dumped were higher, up to 4 mSv/year, which still is of the same order as the average annual natural background dose. Moreover, since any of the proposed remedial actions were estimated to cost several million US$ to implement, remediation was not considered justified on the basis of potentially removing a collective dose of 10 man Sv. Doses calculated to marine fauna were insignificant, orders of magnitude below those at which detrimental effects on fauna populations might be expected to occur. Remediation was thus concluded not to be warranted on radiological grounds.

Epstein-Barr virus induces Fas (CD95) in T cells and Fas ligand in B cells leading to T-cell apoptosis.

Epstein-Barr virus (EBV) acute infectious mononucleosis (AIM) is characterized by transient immunosuppression in vivo and increased T-cell apoptosis after ex vivo culture of AIM peripheral blood mononuclear cells. We undertook experiments to test whether EBV or purified virion envelope glycoprotein gp350 could contribute to Fas-mediated T-cell apoptosis. Our in vitro results indicate that EBV increased Fas expression in CD4(+) T cells and Fas ligand (FasL) expression in B cells and macrophages. Purified gp350 was also shown to significantly increase CD95 expression in CD4(+) T cells. When T-cell CD95 was cross-linked, EBV-stimulated T cells underwent apoptosis. The induction of T-cell CD95 by EBV followed by CD95 cross-linking with anti-CD95 monoclonal antibody resulted in a loss in the number of T cells responding to the T-cell mitogens, anti-CD3 antibody, and interleukin-2. These results indicate that, in addition to serving as a principal ligand for the attachment of virus to target cells, gp350 may also act as an immunomodulatory molecule that promotes T-cell apoptosis.

Intracellular accumulation of detergent-soluble amyloidogenic A beta fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice.

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.

Processing of the Alzheimer's disease amyloid precursor protein in Pichia pastoris: immunodetection of alpha-, beta-, and gamma-secretase products.

betaA4 (Abeta) amyloid peptide, a major component of Alzheimer's disease (AD) plaques, is a proteolytic product of the amyloid precursor protein (APP). Endoproteases, termed beta- and gamma-secretase, release respectively the N- and C-termini of the peptide. APP default secretion involves cleavage within the betaA4 domain by alpha-secretase. To study the conservation of APP processing in lower eukaryotes, the yeast Pichia pastoris was transfected with human APP695 cDNA. In addition to the full-length integral transmembrane protein found in the cell lysate, soluble/secreted APP (sAPP) was detected in the culture medium. Most sAPP comprised the N-terminal moiety of betaA4 and corresponds to sAPPalpha, the product of alpha-secretase. The culture medium also contained minor secreted forms detected by a monoclonal antibody specific for sAPPbeta (the ectodomain released by beta-secretase cleavage). Analysis of the cell lysates with specific antibodies also detected membrane-associated C-terminal fragments corresponding to the products of alpha and beta cleavages. Moreover, immunoprecipitation of the culture medium with three antibodies directed at distinct epitopes of the betaA4 domain yielded a 4 kDa product with the same electrophoretic mobility as betaA4 synthetic peptide. These results suggest that the alpha-, beta-, and gamma-secretase cleavages are conserved in yeast and that P. pastoris may offer an alternative to mammalian cells to identify the proteases involved in the generation of AD betaA4 amyloid.

Secretion of Alzheimer's disease Abeta amyloid peptide by activated human platelets.

Alzheimer's disease (AD) is characterized by the deposition of Abeta (betaA4) peptides of 39 to 43 amino acid residues, which are normal cellular metabolic products derived by proteolysis of the amyloid precursor protein (APP). The physiologic function of Abeta/APP in vivo is poorly understood. We analyzed human platelets for Abeta production by immunoprecipitation coupled to immunoblotting. A 4-kd Abeta fragment that comigrates with an Abeta40 synthetic peptide and reacts with several antibodies specific for the N- and C-termini of Abeta is detected. The majority of platelet Abeta appears to end at residue 40, as determined by immunoreactivity with an Abeta40-specific antibody. Furthermore, Abeta is secreted upon platelet stimulation with the physiologic agonists thrombin and collagen, together with secretion of soluble APP (sAPP). A comparison between serum and plasma shows a 1.6-fold increase in Abeta levels and a 2.4-fold increase in sAPP levels in serum. This is consistent with the view that platelets are the primary source of circulating Abeta and APP. The release of platelet Abeta by physiologic stimuli suggests that it may play a role in platelet aggregation and coagulation or in the repair mechanisms associated with injury.

Anti-interleukin-10 antibodies in patients with chronic active Epstein-Barr virus infection.

The Epstein-Barr virus (EBV) genome encodes a protein in its BamHI C restriction fragment rightward open-reading frame-1 (designated BCRF1 or viral interleukin-10 [vIL-10]) that shares protein homology and biologic properties with human IL-10. Several EBV disorders are characterized by prolonged active EBV infection. Because continued EBV replication could allow for increased vIL-10, ELISA and immunoprecipitation were used to determine whether vIL-10 expression during chronic active EBV infection resulted in vIL-10 and IL-10 antibodies. IL-10 antibodies were assayed in patients diagnosed with chronic and acute infectious mononucleosis (CIM, AIM), nasopharyngeal carcinoma (NPC), and EBV-associated lymphoproliferative disease (LPD), as well as from healthy organ transplant patients and EBV-negative or EBV-positive persons. Whether anti-IL-10 antibodies could inhibit IL-10 biologic activity was determined. vIL-10 antibodies were found in CIM, NPC, and LPD patients and antibodies reactive to IL-10 were found in CIM patients. One CIM patient had IL-10 antibodies that neutralized IL-10 bioactivity in vitro.

Antibody and antibody-dependent cellular cytotoxicity responses against the BamHI A rightward open-reading frame-1 protein of Epstein-Barr virus (EBV) in EBV-associated disorders.

Antibody-dependent cellular cytotoxicity (ADCC) is an important antiviral effector mechanism. ADCC to the protein encoded by the Epstein-Barr virus (EBV) BamHI A rightward open-reading frame-1 (BARF1) was studied by transducing Raji-tk- cells with the BARF1 gene using a retroviral expression vector. The transduced Raji cells expressed BARF1 on the cell surface, as determined by flow cytometry. Sera from chronic and acute infectious mononucleosis and nasopharyngeal carcinoma patients were found to contain antibodies that react with the BARF1 protein. When BARF1-expressing Raji cells were used as targets for ADCC, sera from several nasopharyngeal carcinoma patients demonstrated significant ADCC reactivity, whereas sera from healthy EBV-seronegative and -seropositive persons lacked such reactivity. BARF1-specific ADCC activity could be competitively inhibited with recombinant BARF1 protein. The level of anti-BARF1 antibody activity in sera of patients with EBV-associated diseases suggests that the BARF1 protein may serve as a target on EBV-infected cells for ADCC.

Interactions involving cyclosporine A, interleukin-6, and Epstein-Barr virus lead to the promotion of B-cell lymphoproliferative disease.

Post-transplant patients undergoing prolonged Cyclosporine A (CsA) immunosuppressive therapy were reported to have an increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell out-growth as compared to those cultured without CsA. Peripheral blood mononuclear cells (PBMC), following infection with EBV and CsA treatment, demonstrated increased IL-6 activity in the culture supernatant. The induction of IL-6 appeared to differ within the various lymphocyte populations. In monocytes and B cells, IL-6 expression was preferentially induced by EBV, and initiated by the binding of the two major virion glycoproteins, gp350 and gp220, to CD21, or a CD21-like receptor. Expression of IL-6 in T cells appeared to be due mainly to CsA. B cells also expressed IL-6 following EBV exposure, but not following CsA treatment. EBY-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which was induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. When IL-6 was expressed, it was shown to act as an autocrine growth factor for B cells and to inhibit the immune system allowing for the promotion of B-cell tumors by impairing lymphokine-activated killer cells. Thus CsA treatment, in promoting both increased numbers of lytic EBV B cells and expression of the EBV paracrine growth factor, IL-6, within the microenvironment of EBV B:T cell and EBV B:monocyte interactions, may lead to increased EBV B-cell immortalization and ultimately result in the promotion of B-cell lymphomas in immunosuppressed patients.

Induction of interleukin-6 after stimulation of human B-cell CD21 by Epstein-Barr virus glycoproteins gp350 and gp220.

The cellular receptor for Epstein-Barr virus (EBV) is the type 2 complement receptor, CD21. At initial infection, EBV virion glycoproteins gp350 and gp220 bind to CD21. We report here that the cross-linking of CD21 by gp350/220 results in increased amounts of interleukin 6 (IL-6) RNA and IL-6 protein. This effect could be blocked with anti-gp350/220 and anti-CD21 monoclonal antibodies. Induction of IL-6 in B cells by EBV could be mimicked by treatment with the protein kinase C (PKC) activator phorbol 12,13-dibutyrate but not with the calcium ionophore ionomycin. IL-6 induction by EBV was inhibited with the PKC-specific inhibitor bisindolylmaleimide or the protein tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and herbimycin A, indicating that the induction of IL-6 following CD21 cross-linking is mediated through PKC- and protein tyrosine kinase-dependent pathways.

Interleukin-6 and Epstein-Barr virus induction by cyclosporine A: potential role in lymphoproliferative disease.

Posttransplant patients undergoing prolonged cyclosporine A (CsA) immunosuppressive therapy have been reported to have increased incidence of Epstein-Barr virus (EBV)-associated lymphoproliferative disorders. We undertook experiments to analyze the possible actions of CsA during EBV-infection of human peripheral blood mononuclear cells (PBMC). EBV-infected B cells cultured with CsA demonstrated increased EBV B-cell outgrowth as compared with those cultured without CsA. PBMC, after infection with EBV and CsA treatment, demonstrated increased interleukin-6 (IL-6) activity in the culture supernatant. The induction of IL-6 appears to differ within the various lymphocyte populations. In monocytes, IL-6 expression appears preferentially induced by EBV and is initiated by the binding of the two major virion glycoproteins, gp350 and gp220. Expression of IL-6 in T cells appears to be due mainly to CsA. B cells also express IL-6 after EBV exposure, but not after CsA treatment. EBV-immortalized B-cell lines cultured with CsA exhibited both an increased number of cells expressing viral lytic-cycle antigens and increased amounts of lytic-cycle proteins. IL-6, which is induced by CsA in PBMC, was also capable of inducing the lytic viral cycle in several EBV-immortalized cells. CsA, in promoting both increased numbers of lytic EBV B cells and an EBV paracrine factor, IL-6, within the microenvironment of EBV B cell:T cell and EBV B cell:monocyte interactions, may result in increased EBV B-cell immortalization and ultimately lead to the promotion of B-cell lymphomas in immunosuppressed patients.

Surface expression of the HIV-1 envelope proteins in env gene-transfected CD4-positive human T cell clones: characterization and killing by an antibody-dependent cellular cytotoxic mechanism.

The env gene of the human immunodeficiency virus-type 1 (HIV-1) was transfected in CEM-nkr, a human lymphoid cell line of T lineage that is resistant to the activity of natural killer cells, and for the first time, transfected T cell clones were established that stably express gp160 intracellularly and gp120 on the surface as demonstrated by radioimmunoprecipitation as well as by indirect membrane immunofluorescence. The regulatory protein vpu was not detected by radioimmunoprecipitation in these clones. The surface expression of gp120 without vpu in these clones provides direct evidence that gp160 is processed and cleaved (without vpu) in CD4+ cells. The CD4 antigens of these cells coprecipitated gp160; interestingly, no reduction of the surface CD4 expression (detectable by flow cytometric analysis of membrane immunofluorescence with OKT4) in the transfected cells was observed. However, decreased reactivity of the transfected clones with OKT4A was observed. The gp120-expressing cells did not form syncytia on coculture with other CD4+ human cell lines. These observations suggest the binding of gp120 to the surface CD4 antigen of the transfected cells. The transfected cells retained their resistance to the activity of the natural killer cells but showed a significant (p < 0.05) lysis when they were preincubated with AIDS patients' serum containing anti-gp120/41 antibodies. Thus, the expressed gp120/41 in these cells made them susceptible to killing by an antibody-dependent cellular cytotoxicity (ADCC) mechanism. To our knowledge, these are the first reported CD4+ T cell lines that stably express HIV envelope proteins. These cell lines would be useful as targets in exploring gp120/41-specific immune responses, especially in conducting gp120/41-specific ADCC studies in HIV-infected or gp120/41 (gp160)-vaccinated individuals.