Proline residues in CD28 and the Src homology (SH)3 domain of Lck are required for T cell costimulation.

The Src family tyrosine kinases Lck and Fyn are critical for signaling via the T cell receptor. However, the exact mechanism of their activation is unknown. Recent crystal structures of Src kinases suggest that an important mechanism of kinase activation is via engagement of the Src homology (SH)3 domain by proline-containing sequences. To test this hypothesis, we identified several T cell membrane proteins that contain potential SH3 ligands. Here we demonstrate that Lck and Fyn can be activated by proline motifs in the CD28 and CD2 proteins, respectively. Supporting a role for Lck in CD28 signaling, we demonstrate that CD28 signaling in both transformed and primary T cells requires Lck as well as proline residues in CD28. These data suggest that Lck plays an essential role in CD28 costimulation.

Monoclonal antibodies reactive with distinct domains of the neu oncogene-encoded p185 molecule exert synergistic anti-tumor effects in vivo.

The neu oncogene encodes a 185,000 dalton transmembrane glycoprotein, p185. The current study examined the effects of p185-specific monoclonal antibody administration on the tumorigenic growth of neu-transformed NIH3T3 cells implanted into nude mice. Treatment with anti-p185 monoclonal antibodies of the IgG1, IgG2a, IgG2b subclasses was able to profoundly inhibit the growth of neu-transformed cells. Furthermore, while none of these antibodies individually was able to cause complete eradication of tumors, the administration of mixtures of antibodies reactive with two distinct regions on the p185 molecule resulted in synergistic anti-tumor effects and complete eradication of tumors in a substantial fraction of the treated animals. The effect was oncogene specific, since the growth of ras transformed cells was not influenced by anti p185 antibodies. These results demonstrate that antibodies reactive with multiple domains of a tumor antigen can exert synergistic anti-tumor effects, and suggest that therapy with monoclonal antibodies specific for the neu oncogene product may play a role in cancer treatment.

Monoclonal antibodies specific for the neu oncogene product directly mediate anti-tumor effects in vivo.

We have produced a panel of monoclonal antibodies which bind cell surface domains of the 185 Kd tumor antigen (p185) encoded by the neu oncogene. All of these antibodies stain neu-transformed cells in immunofluorescence assays and immunoprecipitate p185 from metabolically labeled cell lysates. All of the anti-p185 monoclonal antibodies, regardless of isotype, exert a selective cytostatic effect on the growth of neu-transformed cells suspended in soft agar, demonstrating their ability to directly inhibit the transformed phenotype. Anti-p185 antibodies of the IgM, IgG2a, and IgG2b isotypes exert a cytolytic effect on neu-transformed cells in the presence of complement. Only one IgG2a monoclonal antibody is also able to mediate minimal levels of antibody-dependent cellular cytotoxicity (ADCC) (Roussel et al., 1984) in the presence of non-immune spleen cells. In vivo administration of anti-p185 antibodies of the IgG1, IgG2a, and IgG2b isotypes exerts a profound inhibitory effect on the tumorigenic growth of neu-transformed cells. This tumor inhibitory effect is unaffected by depleting tumor bearing animals of complement, and is only minimally affected by depleting tumor bearing animals of macrophages. This suggests that neither complement-mediated killing nor ADCC are necessary for the anti-tumor effects of p185-specific monoclonal antibodies. The results presented here demonstrate that monoclonal antibodies reactive with cell surface domains of an oncogene-encoded protein can directly inhibit tumor growth in vitro and in vivo. Such antibodies may prove useful in the therapy of certain malignancies.

Identification and cloning of a CD43-associated serine/threonine kinase.

CD43 is expressed on most hematopoetic cells and has been shown to regulate the activation and adhesion of T cells. We have cloned a serine/threonine kinase that can interact with the cytoplasmic domain of CD43. This protein is expressed in multiple tissues, including lymphoid cells. Analysis of the subcellular localization reveals it to be present in both the nucleus and the cytoplasm of the cell. The identification of this protein suggests that CD43 may mediate its biologic effects through activation of a kinase cascade, resulting in the regulation of cell growth.

Transfer function comparisons of rat aortic wall image and intravascular pressure harmonic responses.

This study examined the relationship between rat (mature Sprague-Dawley males) thoracic aortic wall and intraluminal pressure responses to a dynamic pressure input. High speed video image (Do) of outer wall area and intravascular pressure (Po) responses of the in vitro aorta were digitized and computer recorded during swept frequency pressure input (2-200 Hz; +/- 10 mm Hg) that was superimposed on static pressures from 20 to 200 mm Hg (Pi). Analysis included Fast Fourier transform (FFT) for Do/Pi and Po/Pi transfer functions and focused on comparison of coefficients from FANSIM (TUTSIM Products) polynomial equation fit to Bode plots for mean data of multiple aortas. The working hypothesis was that Do/Pi = Po/Pi. In FANSIM division by B0 of the general transfer function equality (A1s + A0)/(B2s2 + B1s + B0) yields (a1s + a0)/(b2s2 + b1s + 1); which was the form analyzed. Graphic and statistical comparisons indicated no difference for coefficients a1, a0, b2, and b1 between Do/Pi and Po/Pi. Coefficients b2, and b1 varied with change in level of static pressure. Values for a1 for both Do/Pi and Pi/Po remained relatively constant and appeared independent of static pressure. These results indicated appropriateness of the transfer function form and suggested that: b2, represented inertia of wall and intraluminal fluid mass; b1, represented wall and fluid viscosity influence; a1, represented influence of fluid viscosity and a0, represented influence of wall elasticity.

Intra-arterial pressure wave parameters modeled using electrical analogs.

An Electrical Model was developed to help identify parameters obtained from dynamic pressure data on the in vitro rat aortic artery. The data was obtained using a Multifunction Pressure Generator (MPG) and recording MPG Input Pressure (Pi) and Intraarterial Pressure (Po). Transfer functions of the form Po/Pi = (A1S+Ao)/(B2S2 + B1S+Bo) were obtained and it is necessary to link A1, Ao, B2, B1 and Bo to the Biological Parameters of Inertance (M), Vascular Resistance (R) and Compliance (C). Using the Electrical Analogs to P, M, R and C which are Voltage (V), Inductance (L), Resistance (Re), and Capacitance (Ce), an Electrical Model was built. The Electrical Model has the form Vo/Vi = (Re1S + 1/Ce)/[LS2 + (Re1 + Re2)S + 1/Ce]. Since Ao = Bo = 1 from our experimental data we multiplied the denominator and numerator by Ce to obtain Vo/Vi = (CeRe1S + 1)/[CeLS2 + Ce(Re1 + Re2)S + 1]. We then transformed our Electrical Model to its Pressure Equivalent and obtained Po/Pi = (CR2S + 1)/[CMS2 + C(R1 + R2)S + 1]. Since R2 is less than R1 + R2 we theorize that total R is composed of two viscoelastic or resistive elements R1 and R2. Using measured values of compliance it should be possible to obtain reasonable values for R1, R2 and Inertance.

Comparison of rat aortic, carotid & femoral artery viscoelastic properties using harmonic analysis.

Viscoelastic properties of rat (Wistar Kyota) large (6 aorta), medium (12 carotid) and small (8 femoral) in vitro artery segments, were contrasted over a wide range of static and dynamic pressures. Relationship of change in static pressure (delta dyne/mm2) to diameter (delta mm) was used to estimate a segment's incremental elasticity (KD) at each pressure level. Dynamic intravascular pressure response (Po) was recorded during swept frequency pressure (2-200 Hz; +/- 10 mm Hg) inputs as superimposed on mean pressure steps of 40, 80, 120, 160 and 200 mm Hg (P(i)). Analysis of dynamic data included Fast Fouier Transform of Po/P(i) with FANSIM (TUTSIM Products) curve fit to Bode plots. Curve fit coefficients were used to estimate properties of natural frequency (omega n) damping, viscosity and inertia. Statistical analysis employed ANOVA and SNK multiple comparison procedures. Results indicated that as step-pressure was increased diameter, KD and omega n increased proportionately in all segments. Values of KD and omega n were always highest in femoral and lowest in aortic segments. In all segments damping decreased inversely with increasing pressure while, viscosity and inertia were lowest between 80 and 160 mm Hg. These results documented distinct viscoelastic properties for the three arteries as well as, differences in their response characteristics.

Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen.

The neu oncogene encodes a 185-kDa transmembrane glycoprotein tumor antigen, termed p185. We have recently described a monoclonal antibody reactive with a cell surface domain of the p185 molecule. In vivo treatment with this anti-p185 monoclonal antibody was able to significantly inhibit the tumorigenic growth of neu-transformed NIH 3T3 cells implanted into nude mice. Such treatment had no effect on the tumorigenic growth of Ha-ras-transformed NIH 3T3 cells. Furthermore, anti-p185 antibody treatment was able to inhibit the growth of the rat neuroblastoma cells from which the neu oncogene was initially isolated. These results demonstrate that a monoclonal antibody reactive with the extracellular domain of an oncogene-encoded protein can exert a significant antitumor effect; such antibodies may prove useful in the therapy of certain malignancies.

Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies.

Exposure of neu-oncogene-transformed NIH 3T3 cells to monoclonal antibodies reactive with the neu gene product, p185, results in the rapid and reversible loss of both cell-surface and total cellular p185. Although not directly cytotoxic, monoclonal anti-p185 antibody treatment causes neu-transformed NIH 3T3 cells to revert to a nontransformed phenotype, as determined by anchorage-independent growth. Isotype matched control antibodies of an unrelated specificity do not affect p185 levels or colony formation in soft agar by neu-transformed NIH 3T3 cells. Soft agar colony formation by NIH 3T3 cells transformed by ras oncogenes is not affected by anti-p185 antibody treatment. Anchorage-independent growth of cells from the ethylnitrosourea-induced rat neuroblastoma line in which neu was originally detected by DNA transfection is also inhibited in the presence of anti-p185 monoclonal antibodies. Collectively, these results suggest that p185 is required to maintain transformation induced by the neu oncogene.

Monoclonal antibodies identify a cell-surface antigen associated with an activated cellular oncogene.

A variety of antigens have been identified on the surface of the malignant cell. However, identical antigens are often found on non-malignant cells of the same or different histological origin, or of a different stage of embryonic development. Many of these tumour-associated antigens appear to be only incidentally expressed on neoplastic cells. Clearly, it would be of great interest to identify cell-surface antigens whose expression is associated specifically with the transformed state and linked directly with the mechanisms responsible for transformation. The detection of activated cellular oncogenes in human and animal cancer cells by the technique of DNA transfection has allowed the isolation of genetic elements which are thought to have a critical role in malignancy. Here, in an effort to identify cell-surface antigens associated with the neoplastic process, we have generated hybridomas which secrete monoclonal antibodies that react specifically with cell-surface determinants found on NIH 3T3 cells transformed by transfection with a group of rat neuroblastoma oncogenes. These antibodies bind to and immunoprecipitate a phosphoprotein of relative molecular mass 185,000 (185 K) from a DNA donor rat neuroblastoma and 13 independent rat neuroblastoma DNA transfectants. There was no antibody reactivity with normal NIH 3T3 cells or with NIH 3T3 cells transformed by various other agents.

Harmonic analysis of intravascular pressure response as an index of arterial wall mechanical properties.

The ability to routinely assess mechanical properties of large blood vessels, like the aorta, before an aneurysm or rupture occurs, could benefit diagnostic and therapeutic procedures and save lives. In this study, images of the wall area and intravascular pressure (IP) responses of in vitro rat aorta were recorded during swept frequency pressure input (2-200 Hz; +/- 10 mm Hg) superimposed on mean pressures from 20 to 160 mm Hg. Data analysis included Fast Fourier transform (FFT) of input and responses. Wall and IP responses were underdamped with respective resonance frequencies (Wn) that varied as a function of mean input pressure and the nonlinear nature of wall elasticity. Results indicated closely coupled wall and IP responses and suggested that the IP response may be an adequate index of wall elasticity without need of a direct measure of wall displacement. We considered results to be a key step towards development of a clinical tool which would facilitate analysis of mechanical properties of in vivo conducting vessels.

Synchronizing blood vessel response CCD imagery to swept frequency dynamic pressure signals.

Harmonic analysis of the pressure and wall responses of a blood vessel exposed to a dynamic pressure input signal required the development of a software application which could properly synchronize the data gathered by two separate microcomputers. In order to accomplish this task, the Pressure-Image Editor was developed. The first computer is used to generate a swept frequency sinusoidal dynamic pressure input signal while at the same time monitoring the resulting response pressures. The second computer is used to record the physical (visual) response of the artery to the pressure signal via a high speed CCD camera and video digitizer. Using the Pressure-Image Editor, 256 animated images along with 65,536 pressure points can be combined and synchronized based on the camera frame rate, input trigger frequency, and any internal timing delays. The Pressure-Image Editor is a object-oriented application written in C++ and includes a window based graphical user interface.