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This work provides a novel framework for identifying coma and brain death consciousness states by analysing frequency power and phase synchrony features from electroencephalogram (EEG). The proposed analysis of pairs of EEG electrodes using complex extensions of Empirical Mode Decomposition (EMD) permits the extraction of information related to the state of the brain function. Analysis on 34 subjects in the coma and quasi-brain-death states suggests that phase synchrony constitutes a feasible feature to discriminate quasi-brain-death from coma state. Thus, illustrate the effectiveness of the proposed methods for brain consciousness identification. The predictive power of the features extracted is evaluated by building classification models using support vector machine (SVM) and evaluation the models through receiver operating characteristic (ROC) analysis.

In order to implement affective computing, there have been several studies to elicit human emotion using audio and video stimuli or by recalling previous events. Taste-elicited emotion has also been investigated using food to induce different levels of pleasure. This is monitored using a range of methods, from questionnaire feedback to electrophysiological responses of autonomic nervous system (ANS) and central nervous system (CNS). In this work, we establish that emotions elicited by taste can be monitored using electroencephalogram (EEG), and, for rigour, compare the response to a taste stimulus against the response to the recall of the same taste. The character of emotions were assessed using a subjective measurement, the hedonic score, which describes the pleasant or unpleasant moods of subjects in response to each taste. The classification performance of EEG responses shows excellent separability between the different emotions induced by different tastes. In addition, it is shown that emotion elicited by taste recall is stronger than the stimulus-elicited emotion.

In recent years, many alternative methodologies and techniques have been proposed to perform nondestructive inspection and maintenance operations of moving structures. In particular, ultrasonic techniques have shown to be very promising for automatic inspection systems. From the literature, it is evident that the neural paradigms are considered, by now, the best choice to automatically classify ultrasound data. At the same time, the most appropriate preprocessing technique is still undecided. The aim of this paper is to propose a new and innovative data preprocessing technique that converts real-valued ultrasonic data into complex-valued signals. This allows analysis using phase synchrony, a robust tool that has been previously employed in brain science for establishing robust features in noisy data. Synchrony estimation is achieved using complex extensions of empirical mode decomposition, a data-driven algorithm for detecting temporal scales, thus facilitating the modeling of nonlinear and nonstationary signal dynamics. Experimental tests aiming to detect defective areas in composite materials are reported, and the effectiveness of the proposed methodology is illustrated.

The operation of the noise-assisted multivariate empirical mode decomposition (NA-MEMD) algorithm, which represents a breakthrough in data-adaptive analysis, is illustrated for the time-frequency analysis of Doppler radar signals. The performance of the NA-MEMD is here compared to the continuous wavelet transform, for both synthetic and real-world data applications, showing the advantage of the noise-assisted concept in terms of sparse time-frequency localization. For the considered application, we show how the approach gives a clear and natural interpretation of Doppler radar phenomena and enables the accurate tracking of object speeds. (4 pages)

We reported previously that human CD4+ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-1 viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HIV-1-infected patients in vivo.

A panel of retinoid compounds (tretinoin, isotretinoin, acitretin, and RO13-1470) were tested for inhibitory activity against Kaposi's sarcoma cell (KSC) cultures in vitro. Tretinoin was found to be the most effective retinoid tested, inhibiting the growth of KSC in vitro while having no effect on the expression of interleukin-6 and basic fibroblast growth factor, two important cytokines involved in KSC growth. Tretinoin also did not appear to downregulate the expression of receptors for these two cytokines. At low concentrations (10(-9) M), acitretin and tretinoin selectively inhibited growth of early passage KSC. At higher concentrations (10(-6)-10(-5) M), retinoid treatment induced a pattern of DNA degradation and morphological changes in KSC characteristic of apoptosis (programmed cell death). The inhibitory activity of tretinoin on KSC growth was decreased if human serum (but not fetal calf serum) was present in the growth medium, and partially restored by removal of serum lipids. These data suggest that retinoids possess potential as therapeutic agents in Kaposi's sarcoma.

A novel method is introduced to determine asymmetry, the lateralization of brain activity, using extension of the algorithm empirical mode decomposition (EMD). The localized and adaptive nature of EMD make it highly suitable for estimating amplitude information across frequency for nonlinear and nonstationary data. Analysis illustrates how bivariate extension of EMD (BEMD) facilitates enhanced spectrum estimation for multichannel recordings that contain similar signal components, a realistic assumption in electroencephalography (EEG). It is shown how this property can be used to obtain a more accurate estimate of the marginalized spectrum, critical for the localized calculation of amplitude asymmetry in frequency. Simulations on synthetic data sets and feature estimation for a brain-computer interface (BCI) application are used to validate the proposed asymmetry estimation methodology.

In recent years many alternative methodologies and techniques have been proposed to perform non-destructive inspection and maintenance operations of moving structures. In particular, ultrasonic techniques have shown to be very promising for automatic inspection systems. From the literature, it is evident that neural paradigms are considered, by now, the best choice to automatically classify ultrasound data. At the same time the most appropriate pre-processing technique is still undecided. The aim of this paper is to propose a new and innovative data pre-processing technique that converts real-valued ultrasonic signals into complex-valued signals, making it suitable to apply phase synchrony analysis using complex extensions of Empirical Mode Decomposition (EMD), a data driven algorithm for detecting temporal scales in nonlinear and nonstationary data. Experimental tests aiming to detect defective areas in aircraft components are reported and the effectiveness of the proposed methodology is demonstrated.

This chapter contains sections titled: Introduction Data Fusion Via Fission Brain Death Detection EEG Data Empirical Mode Decomposition Conclusion References

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.

The Empirical mode decomposition (EMD) algorithm is a fully data-driven method which is used to perform an adaptive decomposition of nonlinear and nonstationary signals. It has been recently illustrated that its complex extensions can be used to carry out fusion of multiple images. This is possible because the complex EMD allows comparison between common frequency scales, by aligning them within a single complex IMF. In this paper, complex extensions of EMD are proposed for the fusion of two images; the fusion methodologies are presented for both gray-level and RGB based color images. The potential of the proposed scheme is highlighted by showing its superiority to wavelet based fusion schemes, through simulations on real world multi-exposure images.

This study aims at providing signal processing solutions for the conditioning of multimodal information in audio-aided smart camera environments. A novel approach is introduced for processing audio and video within a unified ‘data fusion via fission’ framework. This is achieved using empirical mode decomposition (EMD), a fully data-driven algorithm which facilitates analysis at multiple time-frequency scales. Its adaptive nature makes it suitable for processing real-world data and allows, for example, signal conditioning (denoising, illumination invariant video) and robust feature extraction. Furthermore, complex extension of the EMD algorithm are used to quantify shared dynamics between the conditioned modalities facilitating multimodal fusion. The proposed collaborative approach is used to model human-human interaction.

In response to a difference in pricing, the San Diego Veterans Administration Medical Center changed its tuberculin preparation from Tubersol to Aplisol in the fall of 2006. Following the change, an increased number of employee skin test conversions was noted. Employee tuberculin skin test converters from 2006 were screened with the QuantiFERON Gold (QFT-G) gamma interferon release assay. Those employees who tested negative by QFT-G were asked to repeat their skin test with both Tubersol and Aplisol tuberculin preparations. Of the new purified protein derivative converters, 12 of 14 returned for repeat testing with QFT-G, and the assay was negative for 83% (10/12), positive for 8% (1/12), and indeterminate for 8% (1/12) of the individuals. Nine of the individuals who were QFT-G negative agreed to repeat skin testing with both tuberculin preparations, and 7/8 (87.5%) demonstrated reactivity with the Aplisol preparation, while 0/8 (0%) reacted to the Tubersol preparation. A change from Tubersol to Aplisol resulted in elevated tuberculin skin test conversion rates that may be due to false-positive reactions. The differences in skin test reactivity between preparations support CDC guidelines that recommend that institutions should not change tuberculin preparations, as doing so may falsely increase the number of positive reactions.

A titer for homologous viral neutralization activity (greater than 1:19,683) was observed after a 3.5-year immunization period with an octameric, branching peptide representing the principal neutralizing determinant (PND) of the human immunodeficiency virus-1IIIB envelope protein. Booster immunizations elicited persistent and potent antibodies in guinea pigs, exceeding responses produced by a conventional bovine serum albumin conjugate by 100-fold. Peptide length, central presentation of a conserved sequence, and inclusion of an upstream sequence contributed to immunogenicity. Titers (greater than 1:1,000) of heterotypic neutralizing antibodies also developed. Octameric PND peptides are a promising approach for an acquired immunodeficiency syndrome (AIDS) vaccine.

SUMMARY An investigation was undertaken to determine whether a recombinant gp 160 envelope protein, which is currently being evaluated as a vaccine for AIDS, induces or modulates the production of tumour necrosis factor-alpha (TNF-α) or interleukin-1β (IL-1β). Incubation of monocytes from healthy, HIV-seronegative persons with 0.0001–1.0 μg of the recombinant vaccine did not result in the secretion of TNF-α or IL-1β, nor did the recombinant product augment or suppress monokine production by lipopolysaccharide (LPS) stimulated monocytes. The vaccine was also without a stimulatory or modulatory effect upon TNF-α or IL-1β secretion by monocytes from a patient with the AIDS-related complex (ARC) and from the monocytic THP-1 cell line. The lack of effect of gp 160 on monokine production has important implications for its efficacy as a vaccine for AIDS.

Electroencephalogram (EEG) provides a non-invasive way to analyze brain activity. Blinking and movement of the eyes causes a strong electrical activity that can contaminate EEG recordings, particularly around the forehead but also as far as in occipital areas. Removal of such ocular artifacts is a considerable signal processing problem, since those artifacts overlap in frequency domain with EEG. In this paper we propose a signal reconstruction method based on a time frequency analysis tool known as the Hilbert-Huang spectrum. We demonstrate how our reconstruction scheme can be successfully applied to contaminated EEG data for the purposes of removing unwanted ocular artifacts.

We assessed the effect of herpes simplex virus type 2 (HSV-2) acquisition on the plasma HIV RNA and CD4 cell levels among individuals with primary HIV infection using a retrospective cohort analysis. We studied 119 adult, antiretroviral-naive, recently HIV-infected men with a negative HSV-2-specific enzyme immunoassay (EIA) result at enrollment. HSV-2 acquisition was determined by seroconversion on HSV-2 EIA, confirmed by Western blot analysis. Ten men acquired HSV-2 infection a median of 1.3 years after HIV infection (HSV-2 incidence rate of 7.4 per 100 person-years of follow-up). The median time of follow-up after acquiring HSV-2 infection was 303 days. All men except 1 were asymptomatic during HSV-2 acquisition, and only 1 HSV-2 seroconverter, who was asymptomatic, had a transient increase in blood HIV load (0.5 log10 copies/mL over 11 days). The HSV-2 incidence rate was high in our cohort of recently HIV-infected individuals; however, HSV-2 acquisition did not significantly change the plasma HIV dynamics and CD4 cell levels.

Information “fusion” via signal “fission” is addressed in the framework of Empirical Mode Decomposition (EMD). In this way, a general nonlinear and non stationary signal is first decomposed into its oscillatory components (fission); the components of interest are then combined in an ad hoc or automated fashion in order to provide greater knowledge about a process in hand (fusion). The extension to the field of complex numbers C is particularly important for the analysis of phase-dependent processes, such as those coming from sensor arrays. This allows us to combine the data driven nature of EMD with the power of complex algebra to model amplitude-phase relationships within multichannel data. The analysis shows that the extensions of EMD to C are not straightforward and that they critically depend on the criterion for finding local extrema within a complex signal. For rigour, convergence of EMD is addressed within the framework of fixed point theory. Simulation examples on information fusion for brain computer interface (BCI) support the analysis.

Retroviral vectors have been developed which produce a secreted form of the helper/inducer T-cell antigen, CD4. Amphotropically packaged vectors were used to transduce cells, and these cells were shown to express the secreted CD4 (sCD4) gene product. The sCD4 produced by the viral vectors is immunoprecipitated by monoclonal antibodies against CD4, which specifically block human immunodeficiency virus (HIV) infection of helper/inducer T cells. A direct physical interaction of vector-produced sCD4 and HIV-1 gp120 was demonstrated by coprecipitation of sCD4/gp120 with antiserum directed against HIV gp120. Furthermore, transduced cells producing sCD4 can protect HIV-susceptible cells from infection by HIV. These data suggest that gene therapy is a potential approach for the treatment of AIDS.

Vascular smooth muscle cells (VSMCs) undergo phenotypic modulation, changing from a differentiated, contractile to a de-differentiated, synthetic phenotype; the change is associated with decreased expression of smooth muscle (SM)-specific genes and loss of cGMP-dependent protein kinase (PKG), but transfection of PKG into de-differentiated VSMCs restores SM-specific gene expression. We show that small interference RNA-mediated down-regulation or pharmacologic inhibition of PKG reduced SM-specific gene expression in differentiated VSMCs and provide a mechanism for cGMP/PKG regulation of SM-specific genes involving the cysteine-rich LIM-only protein CRP4. PKG associated with CRP4 and phosphorylated the protein in intact cells. CRP4 had no intrinsic transcriptional activity, but exhibited adaptor function, because it acted synergistically with serum response factor (SRF) and GATA6 to activate the SM-alpha-actin promoter. cGMP stimulation of the promoter required PKG and CRP4 co-expression with SRF and GATA6. A phosphorylation-deficient mutant CRP4 and a CRP4 deletion mutant deficient in PKG binding did not support cGMP/PKG stimulation of the SM-alpha-actin promoter. In the presence of wild-type but not mutant CRP4, cGMP/PKG enhanced SRF binding to a probe encoding the distal SM-alpha-actin promoter CArG (CC(AT)(6)GG) element. CRP4 and SRF associated with CArG elements of endogenous SM-specific genes in intact chromatin. Small interference RNA-mediated down-regulation of CRP4 prevented the positive effects of cGMP/PKG on SM-specific gene expression. In the presence of CRP4, cGMP/PKG increased SRF- and GATA6-dependent expression of endogenous SM-specific genes in pluripotent 10T1/2 cells. Thus, CRP4 mediates cGMP/PKG stimulation of SM-specific gene expression, and PKG plays an important role in regulating the phenotype of VSMCs.

A robust and stable method for denoising images is proposed based on a novel sampling scheme and the recently developed generalized normalized gradient descent (GNGD) algorithm. The robustness of GNGD is suited for the proposed strategy which, using prior knowledge, resamples input data in order of decreasing (increasing) value before applying the adaptive algorithm. The approach facilitates a high level of denoising ability without causing the filtered data to experience unwanted distortion effects that afflict similar filtering algorithms. The performance of the approach is demonstrated by denoising simulations and comparison with other adaptive filter algorithms. Image data is used in simulations to demonstrate visually how distortion effects are kept to a minimum.

Short interfering RNAs (siRNAs) targeting HIV-1gag, vif, tat, rev and host CD4 and CCR5 have been reported to inhibit HIV replication. However, the sequence divergence of HIV and the concentration dependence of siRNA activity represent significant challenges to RNAi mediated inhibition. To determine the parameters of RNAi in suppression of HIV-1 we screened seven siRNA candidates targeting highly conserved regions of gag/pol, based on target site GC content, for antiviral activity at varying concentrations. Only two of these inhibited CA-p24 production more than 50%, 2064 and 2161. Activity varied with concentration, with 100 nM producing optimal suppression. Requirements for target sequence conservation and activity over a range of concentrations may severely limit the number of siRNA candidates for therapeutic development.

Small interfering RNA (siRNA) and microRNA silence genes at the transcriptional, posttranscriptional, and/or translational level. Using human tissue culture cells, we show that promoter-directed siRNA inhibits transcription of an integrated, proviral, elongation factor 1alpha (EF1A) promoter–green fluorescent protein reporter gene and of endogenous EF1A. Silencing was associated with DNA methylation of the targeted sequence, and it required either active transport of siRNA into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction. These results demonstrate that siRNA-directed transcriptional silencing is conserved in mammals, providing a means to inhibit mammalian gene function.

Among non-primate vertebrates, feline immunodeficiency virus (FIV) infection in the cat may be the closest model of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS). Clinical evolution and immunological and virological relationships between human HIV/AIDS and disease produced by FIV infection in cats are very close. These similarities should facilitate progress in the understanding of mechanisms of viral infection and immunopathology, and make this model potentially very valuable in evaluation of experimental therapeutic approaches to AIDS in man. Development of feline immunodeficiency virus vectors bearing therapeutic genes targeting different human diseases is a promising strategy for gene therapy, despite some recent studies which suggest that despite lack of evidence of infection of man by FIV, additional epidemiological surveillance may be indicated to determine if transmission can occur from this close companion to humans in some circumstances.

To evaluate the efficacy and safety of topical alitretinoin gel (9-cis-retinoic acid [LGD1057], Panretin gel; Ligand Pharmaceuticals, Inc, San Diego, Calif) in cutaneous Kaposi sarcoma (KS).Open-label, within-patient, controlled, dose-escalating phase 1 and 2 clinical trials. In all patients, 1 or more cutaneous KS lesions were treated with alitretinoin gel, and at least 2 other lesions served as untreated controls for up to 16 weeks. Alitretinoin (0.05% or 0.1% gel) was applied twice daily for the first 2 weeks and up to 4 times daily thereafter, if tolerated.Nine academic clinical centers.One hundred fifteen patients with biopsy-proven acquired immunodeficiency syndrome (AIDS)-related KS.AIDS Clinical Trials Group response criteria.Statistically significant clinical responses were observed in 31 (27%) of 115 patients for the group of treated index lesions compared with 13 (11%) for the group of untreated control lesions (P.001). Responses occurred with low CD4(+) lymphocyte counts (200 cells/microL) and in some patients with refractory response to previous systemic anti-KS therapy. The incidence of disease progression was significantly lower for treated index lesions compared with untreated control lesions (39/115 [34%] vs 53/115 [46%]; P =.02). Alitretinoin gel generally was well tolerated, with 90% of treatment-related adverse events confined to the application site and only mild or moderate in severity.Alitretinoin gel has significant antitumor activity as a topical treatment for AIDS-related KS lesions, substantially reduces the incidence of disease progression in treated lesions, and is generally well tolerated.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Gene therapy is a promising treatment modality for acquired immunodeficiency syndrome (AIDS). Autologous transplantation with genetically altered pluripotent hematopoietic stem cells encoding anti-human immunodeficiency virus (HIV) genes could in theory completely and permanently reconstitute all blood lineages and immune functions with cells resistant to HIV. Recent studies showed that CD34+ stem cell can be mobilized in HIV-infected individuals after granulocyte colony-stimulating factor (G-CSF) administration without major side effects or increase of viral load. In this study, peripheral blood CD34+ cells of five HIV-infected individuals were mobilized with G-CSF and after leukapheresis and enrichment, subjected to retroviral transduction with genes encoding anti-HIV ribozyme-decoy fusion molecules. These cells were tested for the ability to give rise to progeny cells, for retroviral transduction efficiency, and for expression of the transgene. CD34+-derived macrophage-like cells were also challenged with HIV. Results showed that CD34+ cells from HIV-infected individuals gave rise to similar numbers of progeny colonies as cells from healthy donors. The transduction efficiency of these cells varied from 68.8 to 100% as assessed by DNA polymerase chain reaction (PCR) of the transgene in individual colonies. CD34+-derived macrophages expressed anti-HIV genes and displayed a substantial and sustained inhibition of HIV replication as compared to untransduced cells. Furthermore, we showed that after thawing, cryopreserved CD34+ cells from these individuals have survival, proliferation, and transduction parameters comparable to fresh cells. Thus, CD34+ cells from HIV-infected patients can be stored for further genetic manipulations with improved vectors or anti-HIV genes as they become available.

Minimal vectors derived from the feline immunodeficiency virus (FIV) were constructed which retained only 259 bp of gag cds, the FIV RRE, and incorporated the woodchuck posttranscriptional regulatory element (wPRE), human immunodeficiency virus (HIV) or FIV cPPT/CTS elements, and a variety of eGFP reporter gene promoters (including CMV IE, EF1a, PGK, MSCV, and MPSV). A packaging plasmid (pC34N) deleted of 1569 bp in the env gene and the principal packaging site was used to generate vectors pseudotyped with the VSV-G protein after transfection into 293FT cells. These second generation FIV vectors generated high titers of 106 TU/ml (up to 109 TU/ml upon concentration), and transduced primary human cord blood CD34+ cells at efficiencies of 50–75% (moi 20-40). Expression persisted at the level of 15–20% after 12 days in culture. Vectors containing the EF1a promoter and cPPT /CTS elements transduced CD34+ cells most efficiently. While the FIV LTR is relatively quiescent in human cells (no LTR-driven expression detected in transduced cells expression by RT PCR), "self-inactivating" 3rd generation FIV vectors were constructed by progressive deletion of the U3 in the 3'LTR, encompassing TATA boxes and binding sites for NF-kB, AP1 and C/EBP, in an attempt to increase expression. One FIV SIN vector had 2–3 fold increase in titer, higher mean fluorescence intensity and improved transduction efficiency of CD34+ cells. FIV vector transduced CD34+ cells expressed eGFP upon differentiation into myeloid cells. FIV SIN transduced CD34+ cells gave rise to GFP+ colonies which persisted for more than 3 weeks when plated onto methylcellulose for hematopoietic progenitor cell assays. Integration assay (two -step Alu PCR) demonstrated that these vectors were integrated into the genome. FIV SIN vectors have potential as an alternative to HIV-1 based vectors for stem cell gene therapy.

The human immunodeficiency virus type 1 strain MN (HIV-1MN) principal neutralizing determinant (PND, V3 loop) was introduced into infectious molecular clones HIV-2KR and simian immunodeficiency virus mm239 (SIVmm239) by hybridization PCR, replacing the corresponding HIV-2 or SIV envelope cysteine loops with the HIV-1 coding sequence. The HIV-2 chimera (HIV-2KR-MNV3) was found to be capable of infecting a number of T-cell lymphoblastic cell lines as well as primary peripheral blood mononuclear cells. In contrast, the SIV chimera (SIV239MNV3) was not replication competent. Envelope produced by HIV-2KR-MNV3 but not the parental HIV-2KR was recognized by V3-specific and HIV-1-specific polyclonal antisera in radioimmunoprecipitation assays. HIV-2-specific antisera recognized both the chimeric and parental virus but not HIV-1MN. The chimeric HIV-2KR-MNV3 virus proved to be exquisitely susceptible to neutralization by HIV-1-specific and V3-specific antisera, suggesting the potential for use in animal models designed to test HIV-1 vaccine candidates which target the PND.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

To increase the inherently weak immunogenicity of synthetic peptide vaccines, we used recombinant DNA techniques to generate chimeras between immunogenic determinants of human immunodeficiency virus type 1 (HIV-1) gp120 and antibody Fab fragments reactive with surface structures displayed specifically on human antigen-presenting cells (APCs), including surface immunoglobulin D (sIgD) and class II major histocompatibility complex (MHC) molecules. Hybridomas producing anti-human MHC class II (HLA-DR) or surface immunoglobulin D monoclonal antibodies (MAbs) that recognize nonpolymorphic determinants were used to clone chimeric Fab gene fragments by employing an established procedure to generate antigen-binding Fab libraries in phagemid vector pComb3. Molecular and immunochemical analysis indicated that the expected chimeric Fab fragments expressing the HIV-1 epitopes were correctly cloned and expressed in Escherichia coli and retained the binding specificity of the native (hybridoma-derived) MAb. The chimeric Fab fragments targeted the linked HIV-1-derived antigenic determinants to the surface of human APCs in vitro, as evidenced by fluorescence-activated cell sorter analysis. Furthermore, such recombinant immunotargeted HIV-1 peptide antigens demonstrated improved immunogenicity over equivalent nonimmunotargeted control antigens, as shown by their ability to stimulate interleukin-2 production by CD4+ T-helper cells from human donors exposed to HIV-1 antigens. These data suggest that immunotargeting of recombinant peptide antigens via the attached Fab fragments facilitates uptake by human APCs with subsequent access to the MHC class II processing pathway, thereby validating the immunotargeting concept for such recombinant subunit vaccines in an in vitro human system.

Drug susceptibility and mutations in the reverse transcriptase (RT) gene were analyzed with 167 virus isolates from 38 patients treated with nevirapine, a potent nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) RT. Resistant isolates emerged quickly and uniformly in all patients administered nevirapine either as monotherapy or in combination with zidovudine (AZT). Resistance developed as early as 1 week, indicating rapid turnover of the virus population. The development of resistance was associated with the loss of antiviral drug activity as measured by CD4 lymphocyte counts and levels of HIV p24 antigen and RNA in serum. In addition to mutations at amino acid residues 103, 106, and 181 that had been identified by selection in cell culture, mutations at residues 108, 188, and 190 were also found in the patient isolates. Sequences from patient clones documented cocirculating mixtures of populations of different mutants. The most common mutation with monotherapy, tyrosine to cysteine at residue 181, was prevented from emerging by coadministration of AZT, which resulted in the selection of alternative mutations. The observations documented that, under selective drug pressure, the circulating virus population can change rapidly, and many alternative mutants can emerge, often in complex mixtures. The addition of a second RT inhibitor, AZT, significantly altered the pattern of mutations in the circulating population of HIV.

Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the beta-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

Human immunodeficiency virus 2 (HIV-2) ISY and the newly derived HIV-2KR are infectious molecular clones that yield viruses differing markedly in their abilities to infect and/or induce syncytia in various T- and monocytoid-cell lines. Chimeric viruses were constructed from these two viral genomes to localize the genetic determinants of some of these properties. Envelope sequences, particularly those spanning the CD4 binding site, appear to be critical for the ability of HIV-2KR to infect MOLT-4 clone 8 and SupT1 cells and to efficiently infect the H9 cell line. On the other hand, multiple determinants may contribute to cytopathicity (gp41 and nef) in H9 cells and replication efficiency in monocytic (THP-1) cells.

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV.

Ribozymes are RNAs that possess the dual properties of RNA sequence-specific recognition, analogous to conventional antisense molecules, and RNA substrate destruction via site-specific cleavage. The cleavage reaction is catalytic in that more than one substrate molecule is processed per ribozyme molecule. We have designed a hairpin ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence (at nucleotides +111/112 relative to the transcription initiation site). The ribozyme was tested in vitro and gave efficient and specific cleavage of RNA containing the leader sequence. To test the antiviral efficacy of this ribozyme, we have cotransfected into HeLa cells HIV-1 proviral DNA and a plasmid expressing the ribozyme from the human beta-actin promoter. HIV-1 expression was inhibited as measured by p24 antigen levels and reduced Tat activity. The antiviral effect of the ribozyme appears to be specific and results from directed RNA cleavage; activity requires both a target sequence and a functional RNA catalytic center. These results suggest that this HIV-1-directed hairpin ribozyme may be useful as a therapeutic agent.

The V3 loop (residues 303-338) of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope protein represents a principal neutralizing determinant for the virus. An HIV-1 proviral clone containing a mutation in the V3 loop was constructed in which the proline residue at position 313 was changed to an alanine (P313-A). This mutation alters the conserved GPGR sequence that is found in the V3 loop sequences of different HIV-1 isolates. The P313-A clone produced virus particles, which were infectious for a number of T-cell lines including MOLT-4, CEM, and SupT1, but demonstrated a relatively low infectivity on the AA5 B-cell line when compared with wild-type viruses, HTLV-IIIB, HXB2/10 (a chimeric molecular clone), and another mutant virus (Q290-T). V3 loop-specific neutralizing polyclonal sera and the 9284 monoclonal antibody, which recognizes the amino side of the V3 loop sequence, effectively blocked infectivity and syncytia formation of all viruses tested. In contrast, the 0.5 beta monoclonal antibody, which is biologically more potent than 9284 and recognizes a different V3 loop determinant, failed to neutralize the P313-A virus. These results suggest that the proline residue in the relatively conserved GPGR "turn" region of the V3 loop is crucial for recognition by the 0.5 beta antibody. The observed variation in sensitivity of the B-cell line to the P313-A virus may reflect the presence of cell-specific factors which could be important in establishing an HIV-1 infection.

Top of pageAbstract Silencing of expression produced by small interfering RNA (siRNA) directed against different regions of a lentiviral vector transgene expressing enhanced green fluorescent protein (EGFP) driven by the elongation factor 1 alpha (EF1a) promoter 12 was investigated in transfected and transduced 293FT fibroblasts and human peripheral mononuclear cells. SiRNA directed against exonic sequences significantly reduced expression, including one targeting exon 1 (+10 to +30 relative to the transcriptional start site), and one targeting EGFP coding sequences (+1167 to +1185). Unexpectedly, siRNA directed against intron A (+155 to +175 relative to the transcriptional start site) or targeting the EF1a promoter (−106 to −86 and −25 to −5 start) also silenced EGFP expression. Post-transcriptional gene silencing in human cells has been described to be restricted to the cytoplasm 3, and transcriptional gene silencing (TGS) mediated by RNA interference (RNAi) has not previously been shown to occur in mammalian cells. Silencing of expression could not be attributed to reduced vector transduction, non-specific inhibition of transcription, cytotoxicity, initiation from a cryptic promoter, or aberrant splicing of EGFP mRNA, and was confirmed to occur at the level of transcription by nuclear run-on. Endogenous EF1a expression could inhibited by transfecting siRNA using a reagent (MPG)4w which facilitates nuclear entry. Inhibition of expression and methylation of one site in the promoter was reversible with addition of 5′ azacytidine and trichostatin A. Treatment with siRNA targeting intronic sequences and promoter regions can induce transcriptional gene silencing. Access of siRNA to the nucleus may be key to the observed effects.