Optimization & Characterization of Interdigitated Electrodes for Microbial Growth Monitoring.

This study optimally designed and implemented highly sensitive microscale interdigitated electrodes (IDEs) to monitor microorganisms' growth in diverse environments. Gold interdigitated electrodes (AuIDE) with 4 mm×4 mm effective sensing area and varying microscale interdigitate gaps were designed and fabricated. The electrodes were electrically characterized voltametrically. Electrochemical impedance spectroscopy (EIS) measurements were conducted to determine the optimal geometry by observing the impedance spectra of microelectrodes through varying pH and temperature. Furthermore, the sensors sensitivity was evaluated by measuring the impedance properties of a microscale volume of microorganism concentrations in growth media solution.

The EcoChip 2: An Autonomous Sensor Platform for Multimodal Bio-environmental Monitoring of the Northern Habitat.

This paper presents the EcoChip 2, an autonomous multimodal bio-environmental sensor platform for the monitoring of microorganisms in the northern habitat. The EcoChip 2 prototype includes an array of 96-wells for the continuous monitoring of microbiological growth through a multichannel electrochemical impedance analyzer circuit. In addition, the platform includes luminosity, humidity, temperature sensors and monitoring. The developed electronic board uses an ultra-low-power microcontroller unit, a custom power management unit, a low-power wireless ISM-2.45 GHz transceiver, and a flash memory to accumulate and store the sensor data over extended monitoring periods. When a wireless base station is placed within the transmission range of the EcoChip 2, an embedded low-power wireless transceiver transmits the 96-wells impedance data and the other sensor data stored in the flash memory to the user interface. We present the measured performance of the prototype, along with laboratory test results of bacterial growth measurements inside the 96 wells in parallel. We show that the EcoChip 2 can successfully measure the impedances associated with bacterial growth over several hours using an excitation frequency of 2 kHz with power consumption of 114.6 mW under operating mode.

Feature Selection with Conjunctions of Decision Stumps and Learning from Microarray Data.

One of the objectives of designing feature selection learning algorithms is to obtain classifiers that depend on a small number of attributes and have verifiable future performance guarantees. There are few, if any, approaches that successfully address the two goals simultaneously. To the best of our knowledge, such algorithms that give theoretical bounds on the future performance have not been proposed so far in the context of the classification of gene expression data. In this work, we investigate the premise of learning a conjunction (or disjunction) of decision stumps in Occam's Razor, Sample Compression, and PAC-Bayes learning settings for identifying a small subset of attributes that can be used to perform reliable classification tasks. We apply the proposed approaches for gene identification from DNA microarray data and compare our results to those of the well-known successful approaches proposed for the task. We show that our algorithm not only finds hypotheses with a much smaller number of genes while giving competitive classification accuracy but also having tight risk guarantees on future performance, unlike other approaches. The proposed approaches are general and extensible in terms of both designing novel algorithms and application to other domains.

Diminished somatostatin gene expression in individuals with HIV and major depressive disorder.

In analyzing the expression of 15 candidate genes for HIV encephalitis (HIVE) by the presence or absence of major depressive disorder (MDD), we noted significant reductions in the expression of four cytoskeletal genes and somatostatin. Whereas disruption of cytoskeletal genes has been noted in HIVE, dysregulation of somatostatin has not, indicating that dysregulation of somatostatin is part of the molecular pathologic process of MDD in the setting of HIV.

Microarray truths and consequences.

For many, analysis of a microarray experiment starts with a spreadsheet of expression levels. While great attention is duly paid to RNA extraction, preparation and hybridization, relatively little care is devoted to extraction of expression levels from the fluorescent image. By delegating this step to a click of the mouse the exact extraction process is masked and researchers may be unwittingly compromising their data. In this review, we describe the most common mistakes committed on the path from the image to the spreadsheet and their impact on data quality. Remedies are further proposed for most of the popular microarray platforms in use today.

IR imaging using uncooled microcantilever detectors.

Uncooled bimaterial microcantilever detectors were fabricated and used to obtain infrared (IR) images of objects at temperatures ranging from room temperature to a few hundred degrees C. Images were obtained using both single 50 micro m x 50 micro m microcantilever IR detectors and arrays of microcantilever detectors. Thermal radiation from the target object was imaged onto the detector and the resulting temperature change caused microcantilever bending due to the bimaterial effect. This micromechanical bending was measured using two different non-contact optical readout techniques and IR images were obtained. A smaller size (20 micro m x 20 micro m) microcantilever IR detector was also used to capture IR images of near room temperature objects.

Feasibility of tunable MEMS photonic crystal devices.

Periodic photonic crystal structures channel electromagnetic waves much as semiconductors/quantum wells channel electrons. Photonic bandgap crystals (PBC) are fabricated by arranging sub-wavelength alternating materials with high and low dielectric constants to produce a desired effective bandgap. Photons with energy within this bandgap cannot propagate through the structure. This property has made these structures useful for microwave applications such as frequency-selective surfaces, narrowband filters, and antenna substrates when the dimensions are on the order of millimeters. They are also potentially very useful, albeit much more difficult to fabricate, in the visible/near-infrared region for various applications when the smallest dimensions are at the edge of current micro-lithography fabrication tools. We micro-fabricated suspended free standing micro-structure bridge waveguides to serve as substrates for PBC features. These micro-bridges were fabricated onto commercial silicon-on-insulator wafers. Nanoscale periodic features were fabricated onto these micro-structure bridges to form a tunable system. When this combined structure is perturbed, such as mechanical deflection of the suspended composite structure at resonance, there can be a realtime shift in the material effective bandgap due to slight geometric alterations due to the induced mechanical stress. Extremely high resonance frequencies/device speeds are possible with these very small dimension MEMS.

Statistical analysis of high-density oligonucleotide arrays: a multiplicative noise model.

MOTIVATION: High-density oligonucleotide arrays (GeneChip, Affymetrix, Santa Clara, CA) have become a standard research tool in many areas of biomedical research. They quantitatively monitor the expression of thousands of genes simultaneously by measuring fluorescence from gene-specific targets or probes. The relationship between signal intensities and transcript abundance as well as normalization issues have been the focus of much recent attention (Hill et al., 2001; Chudin et al., 2002; Naef et al., 2002a). It is desirable that a researcher has the best possible analytical tools to make the most of the information that this powerful technology has to offer. At present there are three analytical methods available: the newly released Affymetrix Microarray Suite 5.0 (AMS) software that accompanies the GeneChip product, the method of Li and Wong (LW; Li and Wong, 2001), and the method of Naef et al. (FN; Naef et al., 2001). The AMS method is tailored for analysis of a single microarray, and can therefore be used with any experimental design. The LW method on the other hand depends on a large number of microarrays in an experiment and cannot be used for an isolated microarray, and the FN method is particular to paired microarrays, such as resulting from an experiment in which each 'treatment' sample has a corresponding 'control' sample. Our focus is on analysis of experiments in which there is a series of samples. In this case only the AMS, LW, and the method described in this paper can be used. The present method is model-based, like the LW method, but assumes multiplicative not additive noise, and employs elimination of statistically significant outliers for improved results. Unlike LW and AMS, we do not assume probe-specific background (measured by the so-called mismatch probes). Rather, we assume uniform background, whose level is estimated using both the mismatch and perfect match probe intensities. RESULTS: We present a new method for GeneChip analysis, based on a statistical model with multiplicative noise. We demonstrated that this method yields results superior to those obtained by the Affymetrix Microarray Suite 5.0 software and to those obtained by the model-based method of Li and Wong (Li and Wong, 2001). The present method eliminates the hard-to-interpret negative expression indices, and the binary 'presence' calls (present or absent) are replaced by the statistical significance (p-value) of gene expression. We have found that thresholding the p-values at the (0.1)(16)-level produces about the same number of 'present' calls as the AMS software. By testing our method on a pair of replicate GeneChips (hybridized with the same cRNA), we found that 95.6% of data points lie within the 1.25-fold interval. In other words, our method had a 4.4% type I error rate at the 1.25-fold level. The error rate of the LW method was 15%, and that of the AMS method was 29%. There were no points outside the 2-fold interval with the present method. Analysis of variance (ANOVA) of another experiment with multiple replicates shows that this reduction of variance is not accompanied by a corresponding reduction of signal. On the contrary, the signal-to-noise ratio (as measured by the distribution of F-statistics) of the present method is on average 3.4-times better than that of AMS, and 1.4-times better than that of Li and Wong.

Role of CD95-activated caspase-1 processing of IL-1beta in TCR-mediated proliferation of HIV-infected CD4(+) T cells.

CD95 plays a critical role in the homeostasis of the immune system, and has been reported to participate in T cell death during HIV infection. Here we report that the response to CD3-TCR stimulation of CD4(+) T cells from HIV-infected individuals and CD4(+) T cells from healthy donors incubated in vitro with HIV-1(Lai) depends on the manner the CD3-TCR complex is engaged. While stimulation by anti-CD3 antibodies in solution induced CD4 T cell apoptosis both in the absence or presence of anti-CD95 antibodies, stimulation by immobilized anti-CD3 antibodies rendered CD4(+) T cells resistant to CD95-mediated death and led to increased CD4 T cell proliferation in response to CD95 ligation. CD95 ligation of CD4(+) T cells led to the activation of caspases, while costimulation induced by anti-CD3 and anti-CD95 mAb prevented the full processing of caspase-3 and caspase-8. Proliferation of CD4(+) T cells induced by CD3-TCR and CD95 costimulation was decreased by treatments with a caspase-1 inhibitor or with neutralizing antibodies to IL-1ss, indicating a requirement for caspase-1-mediated IL-1beta processing and secretion. Our findings suggest a novel mechanism whereby in addition to its role in inducing T cell apoptosis, CD95 signaling during HIV infection may also provide a costimulatory signal leading to an enhancement of CD4 T cell proliferation in response to CD3-TCR complex engagement.

Intracellular delivery of proteins with a new lipid-mediated delivery system.

There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.

Temporal gene regulation during HIV-1 infection of human CD4+ T cells.

CD4(+) T-cell depletion is a characteristic of human immunodeficiency virus type 1 (HIV-1) infection. In this study, modulation of mRNA expression of 6800 genes was monitored simultaneously at eight time points in a CD4(+) T-cell line (CEM-GFP) during HIV infection. The responses to infection included: (1) >30% decrease at 72 h after infection in overall host-cell production of monitored mRNA synthesis, with the replacement of host-cell mRNA by viral mRNA, (2) suppression of the expression of selected mitochondrial and DNA repair gene transcripts, (3) increased expression of the proapoptotic gene and its gene p53-induced product Bax, and (4) activation of caspases 2, 3, and 9. The intense HIV-1 transcription resulted in the repression of much cellular RNA expression and was associated with the induction of apoptosis of infected cells but not bystander cells. This choreographed host gene response indicated that the subversion of the cell transcriptional machinery for the purpose of HIV-1 replication is akin to genotoxic stress and represents a major factor leading to HIV-induced apoptosis.

Use of keyword hierarchies to interpret gene expression patterns.

MOTIVATION: High-density microarray technology permits the quantitative and simultaneous monitoring of thousands of genes. The interpretation challenge is to extract relevant information from this large amount of data. A growing variety of statistical analysis approaches are available to identify clusters of genes that share common expression characteristics, but provide no information regarding the biological similarities of genes within clusters. The published literature provides a potential source of information to assist in interpretation of clustering results. RESULTS: We describe a data mining method that uses indexing terms ('keywords') from the published literature linked to specific genes to present a view of the conceptual similarity of genes within a cluster or group of interest. The method takes advantage of the hierarchical nature of Medical Subject Headings used to index citations in the MEDLINE database, and the registry numbers applied to enzymes.

HIV induces lymphocyte apoptosis by a p53-initiated, mitochondrial-mediated mechanism.

HIV-1 induces apoptosis and leads to CD4+ T-lymphocyte depletion in humans. It is still unclear whether HIV-1 kills infected cells directly or indirectly. To elucidate the mechanisms of HIV-1-induced apoptosis, we infected human CD4+ T cells with HIV-1. Enzymatic analysis with fluorometric substrates showed that caspase 2, 3, and 9 were activated in CD4+ T cells with peak levels 48 h after infection. Immunoblotting analysis confirmed the cleavage of pro-caspase 3 and 9, and of specific caspase substrates. Release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria was observed in HIV-infected cells. The cytochrome c and AIF release preceded the reduction of the mitochondrial transmembrane potential and nuclear chromatin condensation. H IV infection led to phosphorylation of p53 at the Ser15 residue, detectable as early as 24 h after infection. The p53 phosphorylation was followed by increased mRNA and protein expression of p21, Bax, HDM2, and p53. Up-regulation of surface FasL expression, accompanied by a down-regulation of Fas-associated proteins (FADD, DAXX, and RIP), was observed 72 h after infection. Our results suggest that HIV activates the p53 pathway, leading to cytochrome c and AIF release with ensuing caspase activation.

Conjugation of protein to immunostimulatory DNA results in a rapid, long-lasting and potent induction of cell-mediated and humoral immunity.

Immunostimulatory DNA sequences (ISS) are a potent Th1 adjuvant. We hypothesized that conjugation of ISS to protein antigens would strongly enhance their immunogenicity because both antigen and adjuvant (ISS) would be delivered to the same locale/antigen-presenting cell. To test this hypothesis, we conjugated a 22-mer immunostimulatory oligodeoxynucleotide (ISS-ODN) to two test antigens of differing intrinsic immunogenicity, namely Escherichia coli beta-galactosidase and the HIV-1 envelope glycoprotein gp120. We show that the antigen-ISS conjugates rapidly induce Th1 cells secreting high levels of IFN-gamma, strong CTL activity, and high titer IgG2a and HIV-neutralizing antibodies, exceeding gene and protein vaccination alone or immunization with mixtures of antigen and ISS-ODN. The data suggest that this procedure generates a novel and unique vaccine that rapidly triggers strong humoral and cell-mediated immunity.

Cutting edge: a novel viral TNF receptor superfamily member in virulent strains of human cytomegalovirus.

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.

Differential expression profiles of apoptosis-affecting genes in HIV-infected cell lines and patient T cells.

OBJECTIVE: To clarify the molecular mechanisms of HIV-induced apoptosis. DESIGN: The assessment of expression patterns for genes affecting the interrelated cell cycle and apoptosis processes in HIV-1LAI-infected T lymph oblastoid (CEM) cells, as well as CD4 and CD8 cells from HIV-infected individuals and controls. METHODS: The kinetics of HIV infection in CEM cells were defined by flow cytometry of green fluorescent protein expression from a reporter vector. Apoptosis of CEM cells was measured by propidium iodine staining and flow cytometry. Gene expression levels were determined by a multiprobe RNase protection assay. RESULTS: The infection and apoptosis of CEM cells were associated with enhanced expression of Bax, Bcl-2, Bcl-X(L) and caspase 1 (ICE). There was increased expression of Bcl-2 and caspase 1 and decreased expression of cyclin-dependent kinase inhibitor p21CIP1 in CD4 cells of HIV-infected individuals compared with uninfected controls. The CD8 cells of HIV-infected individuals exhibited increased expression of Bcl-2, Bcl-X(L), Bax and caspase 1 but, in contrast to the CD4 subset, they showed elevated expression of p21CIP1 and p16INK4a compared with controls. CONCLUSIONS: The Bax increase in CEM cells appears to be a direct effect associated with a high frequency of infection and apoptosis, because it was not found in the CD4 cells of patients. In contrast, the increase of Bax in the CD8 cells of patients seems to be an indirect effect. Increases in Bcl-2, Bcl-X(L) and caspase 1 in HIV-infected CEM cells may be caused by both direct and indirect mechanisms, because they also occurred in CD4 and CD8 cells of HIV-infected individuals. In addition, the low expression of p21CIP1 in the CD4 subset of HIV-infected individuals could promote apoptosis, whereas the high expression of p21CIP1 and p16INK4a in the CD8 subset may lead to a state of anergy, akin to replicative senescence.

Virological importance of the protease-cleavage site in human immunodeficiency virus type 1 Nef is independent of both intravirion processing and CD4 down-regulation.

The HIV-1 Nef protein is present within the virion and is processed there by the viral protease. Mutational analysis indicated that residues 54-60 in HIV-1 Nef were required for intravirion cleavage. When viruses were produced using T cell lines or primary lymphoblasts, these residues were also required for optimal viral infectivity. However, substitution of native Nef residues with those of a functional Gag cleavage site demonstrated that intravirion cleavage was insufficient for the virological function of this domain. Furthermore, the importance of certain cleavage site residues to infectivity was conditional on the producer cell type. In particular, a mutant containing a deletion of residues 54-57 was phenotypically nef defective when produced using T cells (CEM, A2.01, or primary lymphoblasts) but was minimally impaired when produced from 293 or HeLa cells. This mutant was cleavage resistant, indicating that proteolytic processing of Nef was dispensable for infectivity enhancement when virions were assembled in certain non-T cells. Residues 54-61 of the cleavage site, including 54-57, were also required for Nef-mediated down-regulation of CD4. However, the surface expression of CD4 on HeLa cells in amounts comparable to that on the surface of primary T lymphoblasts did not create a producer cell environment in which residues 54-57 acquired greater virological importance. Furthermore, these residues were required for optimal infectivity even during virion assembly in T cells (A2. 01) that expressed a CD4 molecule that is unable to respond to Nef. These data suggested that in producer T cells, certain cleavage site residues (54-57) contribute to a Nef-mediated virological effect that is unlikely to be linked causally to CD4 down-regulation. Conversely, in the context of 293 cells as viral producers, the Delta54-57 mutant separated genetically down-regulation of CD4 (for which it was defective) from enhancement of infectivity (for which it was functional). Together, these data indicate that the virological function of the cleavage site domain is both independent of intravirion proteolytic processing of Nef and independent of CD4 down-regulation.

Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors.

We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF-kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization.

Early modification of host cell gene expression induced by HIV-1.

OBJECTIVE: Characterization of the effects of infection with HIV-1 on cellular gene expression. DESIGN AND METHODS: Differential RNA display was applied to compare uninfected and HIV-1LAI-infected CEM cells 24 h post-inoculation. Differential bands were selected, cloned and several clones per band were sequenced. RNase protection assay was used to confirm differential display findings in HIV-1LAI-infected CEM cells as well as in another T-cell line (H9) infected with a different strain (HIV-1 SF33) RESULTS: Twelve differentially expressed bands, six up- and six downregulated in HIV-infected cells compared with controls, were selected. Four of the six upregulated bands were HIV transcripts. RNase protection assay of the remaining eight bands confirmed differential expression of four genes, including induction of a mariner transposase and moesin as well as suppression of alpha-nascent polypeptide-associated complex and mitochondrial heat shock protein 75 in HIV-1-infected cell cultures. Furthermore, a significant increase of glioma pathogenesis-related protein was found by RNase protection assay. CONCLUSIONS: Based on this initial limited differential display analysis, it was estimated that expression of 3% of the host genes was altered by HIV-1. Amongst the identified gene modifications, the induction of a mariner transposase may alter cellular gene expression itself, whilst the enhanced expression of glioma pathogenesis-related protein suggests a role in the host cell response to viral infection. The increase in moesin may facilitate viral budding and uptake. Furthermore, the suppression of alpha-nascent polypeptide-associated complex may promote translocation of HIV-1 polypeptides into the endoplasmic reticulum, whereas the downregulation of mitochondrial heat shock protein 75 may contribute to a cytopathic effect on mitochondria and possibly impairs antigen presentation.

The Ick protein tyrosine kinase is not involved in antibody-mediated CD4 (CDR3-loop) signal transduction that inhibits HIV-1 transcription.

Monoclonal antibodies (mAb) that bind to the immunoglobulin CDR3-like region in the D1 domain of the CD4 molecule can inhibit the HIV-1 life cycle in CD4-positive T cells and lymphoblastoid cell lines at the stage of transcription. This antiviral effect requires the integrity of the cytoplasmic tail of CD4 which is known to act as a signal transduction region through its association with the protein tyrosine kinase (PTK) p56lck. In this study, we investigated the putative role of this PTK in transducing inhibitory signals that act on HIV-1 replication after triggering by anti-CDR3-like region antibody treatment of infected T cell lines. CEM (CD4+/p56lck + inducible), MT2 (CD4+/p56lck - repressed), HSB-2 (CD4-/p56lck + constitutively), HSB-2 WTCD4 (CD4+/p56lck + constitutively), HSB-2 CD4.402 (CD4+ truncated form which lacks the cytoplasmic domain/p56lck + constitutively), and HSB-2 CD4mut (CD4+ unable to bind lck/p56lck + constitutively) were exposed to HIV-1 and cultured in medium supplemented with an anti-CDR3-like region-specific antibody or a control anti-CD4 mAb which does not inhibit HIV-1 transcription. We found that CDR3-loop-mediated inhibitory signals are efficiently transduced in CD4-positive cells which demonstrate a constitutive activation of p56lck or in CD4-positive cells lacking p56lck expression. Moreover, inhibitory signals were transduced in HSB-2 CD4mut cells expressing a cell surface CD4 with a double cysteine mutation in its cytoplasmic tail that renders the molecule unable to bind p56lck, but not HSB-2 CD4.402 cells expressing a truncated form of CD4 which lacks the cytoplasmic domain. These results indicate that the p56lck plays no direct role in this process and suggests the existence of another signaling partner for CD4.