Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro.

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.

Biochemical and functional analyses of a secreted H-2Ld molecule.

A truncated H-2Ld gene was constructed by deleting the transmembrane and cytoplasmic exons. The truncated H-2Ld gene was introduced into mouse L cells using the thymidine kinase gene as a selectable marker. Transformants were isolated and screened for the presence of truncated H-2Ld antigen. The truncated H-2Ld gene product was present in both the cytoplasm and culture medium, but not on the cell surface. The truncated H-2Ld antigen was stable in culture medium for at least 9 h and was secreted into the medium at a rate similar to the kinetics with which complete H-2 antigens reach the cell surface. Transformants expressing the truncated H-2Ld molecule were not recognized by cytotoxic T lymphocytes specific for the H-2Ld antigen.

Histologic effects of contact ultrasound for the treatment of glaucoma.

The histologic effects of a contact ultrasound applicator were compared with those of the immersion applicator that is currently used clinically for the treatment of glaucoma. The applicator coupling cone uses a distensible rubber membrane that can be inflated to control stand-off distance relative to the surface of the eye. This feature allows the focal point of the therapeutic beam to be placed at selective depths. Histologic comparisons of lesions in rabbit and pig eyes showed lesions in the sclera and ciliary body that were similar to those produced by the immersion transducer when the same focal position was used. Moving the focal point to a greater depth resulted in less superficial damage, yet still produced ciliodestruction. Damage to the blood supply of the ciliary body, as found in human cadaver eyes, may be an additional mechanism of action of therapeutic ultrasound, and perhaps of other transscleral high-energy modalities.

Experimental, ultrasonically induced lesions in the retina, choroid, and sclera.

Lesions of the retina, choroid, and sclera were created in the proptosed eye of the rabbit with the use of focused ultrasound at 9.8 MHz. Exposure levels greatly exceeding those encountered in diagnostic systems were required. Histological examination of selected tissue samples, analysis of lesion-producing exposures, and lesion size variations suggest that thermal mechanisms are the principal cause of permanent tissue alterations. Subthreshold lesions, which consist of transitory effects, were observed. 'Threshold" lesions first manifested tansitory effects followed by permanent alterations which appeared within 24 hr. Suprathreshold lesions were evidenced by the immediate appearance of a highly blanched area with an overlying edema and occasional hemorrhage, appearing within 24 hr. The edema in these cases was resorbed within a few days, leaving behind a permanently blanched region. The threshold curve developed from these data is similar in shape to the threshold curve for light-induced lesions created with a commensurate focal spot.

Proton beam irradiation and hyperthermia. Effects on experimental choroidal melanoma.

Ultrasonically induced hyperthermia (4.75 MHz) and proton irradiation (160 meV) were evaluated alone and combined to treat experimental choroidal melanoma in 58 rabbit eyes. Threshold tumoricidal doses were established for each modality. Therapy was performed combining subthreshold doses of heat and radiation. Focused ultrasonic energy via an external beam was found to deliver well-localized heat to an intraocular tumor. Ectopic temperature elevations due to soft-tissue-bone interfaces were alleviated by modifying beam alignment. The results indicate that hyperthermia (43 degrees C for one hour) potentiated the tumoricidal effects of radiation, while sparing normal ocular structures. Therefore, we believe that experimental hyperthermia is suitable as an adjuvant treatment modality. This shows that ultrasound hyperthermia has the potential to increase the efficacy of proton irradiation by lowering radiation doses and thus decreasing posttreatment ocular morbidity in human intraocular malignancies.

Ultrasonically accelerated resorption of vitreous membranes.

A noninvasive procedure was used to treat membranes within the vitreous with controlled exposure to focused high-intensity ultrasound in rabbit eyes. Membranes were created by injecting small quantities of platelet-rich plasma into the vitreous of the intact globe of anesthetized rabbits. The average time for untreated membranes (controls and shams) to be resorbed was 34 days, whereas the average time to total resorption of membranes treated with ultrasound was 21 days. This comparison showed that high-intensity ultrasound was effective in accelerating the resorption of this type of vitreous membrane. This ultrasound technique is totally noninvasive and can be used without damage to other ocular tissues. Although this animal model departs in some respects from human vitreous membranes, eventual clinical applications are indicated in selected cases where vitrectomy must be delayed or is contraindicated. Noninvasive disruption of vitreous membranes by therapeutic ultrasound offers great promise for reducing the visual limitations produced by such membranes, and eliminating the sequence of disease leading to permanent visual loss.

Ultrasonic hyperthermia and radiation in the management of intraocular malignant melanoma.

Hyperthermia and radiation were used in combination to treat four patients with choroidal malignant melanoma. This technique uses ultrasonically induced hyperthermia synergistically with radiation to destroy tumor cells. The lower levels of radiation used should avoid the late vascular and inflammatory complications seen in conventional radiation therapy. Tumors were scanned by a computerized diagnostic ultrasound system before treatment and assigned an acoustic tissue type on the basis of a statistical comparison of their ultrasound backscatter spectrum with spectra of tumors of known pathologic status. During the follow-up period, the longest of which was 15 months, all tumors demonstrated regression patterns consistent with choroidal tumors of the same acoustic tissue types treated with conventional radiation therapy.

Effect of hyperthermia on experimental choroidal melanoma.

Thirty-five rabbit eyes were implanted subchoroidally with Greene's hamster melanoma. When the tumours reached a base diameter of 5 mm, they were treated with ultrasonically induced hyperthermia with a range of temperatures and exposure durations (43-67 degrees C and 75 s to 60 min). Of the 23 treated eyes examined two months after treatment eight showed complete regression of the tumour. Seven showed initial tumour regression, but there was subsequent regrowth of tumour round the margins of the original mass. In eight eyes the tumour continued to grow, though in some cases the rate of growth appeared to be slower than in the controls. In contrast, in all untreated animals the tumour grew to fill the vitreous cavity. These preliminary findings indicate that ultrasonically induced hyperthermia can be an effective local treatment of this intraocular tumour model.

Treatment of experimental lens capsular tears with intense focused ultrasound.

High-intensity focused ultrasound was employed to seal lens capsular tears in a rabbit model. Ultrasound therapy was applied either contiguously, thereby completely covering the tear, or in a discrete exposure pattern around the tear. Both methods prevented the formation of a generalised cataract. This was in contrast to results observed in a group of control (untreated) animals which all developed generalised lens opacities. Each control animal also developed a local lens opacity at the site of the capsular tear, as did half the animals treated with the discrete pattern. No animal treated with contiguous exposures developed any local or generalised traumatic-type cataract other than the small lens opacity immediately produced by the treatment. These treatment cataracts would not constitute a significant impediment to vision so long as they did not fall on the visual axis.

Production of corneal lesions using high-intensity focused ultrasound.

In order to study the potential use of ultrasound as a noninvasive system for altering corneal curvature, we used high-density focused ultrasound at a frequency of 4.8 MHz and 7.9 MHz to produce corneal lesions in the rabbit eye. Intensity and duration threshold exposure conditions were determined for the production of minimally visible lesions. Threshold lesions were initially apparent as discrete white opacities resulting from stromal edema and disruption. Light and scanning electron microscopy of higher-energy, suprathreshold lesions revealed more extensive disruption, including the formation of a superficial stromal depression and a larger zone of edema and disorganization surrounding each lesion. Posterior stromal lamellae, endothelium, and Descemet's membrane were intact. Healing and reepithelialization resulted in a smooth corneal surface with no residual opacification. Threshold determinations predict safe exposure levels to the cornea during insonification of other ocular structures. Selective heating of the peripheral cornea using focused ultrasound may be a useful technique for correcting astigmatism.

Thermal model for ultrasonic treatment of glaucoma.

Focused ultrasound, at a frequency of 4.6 MHz, was used to create lesions of the sclera in the proptosed glaucomatous eye of the anesthetized albino rabbit, with the result that elevated intra-ocular pressures were reduced to normal levels. Initial trials on carefully selected glaucoma patients have also shown that pressure reductions can be produced with high-intensity ultrasound. A theoretical model was developed to compute the spatio-temporal features of temperature rises induced in the sclera during these treatments. Experimental data confirmed the accuracy of the model.

Computer model of ultrasonic hyperthermia and ablation for ocular tumors using B-mode data.

Computer simulations have been conducted to examine hyperthermia and ablation for treating ocular tumors. An interactive software package has been implemented that permits relevant tissue dimensions to be determined from B-mode data. This package also permits interactive beam positioning, and it provides image displays depicting computed absorbed doses and temperature rises. Results are presented showing how hyperthermia temperature patterns are influenced by beam position, beam geometry and frequency. Images showing ablative temperature rises at various time intervals are also presented. For hyperthermia, geometric models of beam profiles showed that a non-uniform beam pattern (with a central low-intensity region) can produce more uniform heating of small ocular tumors than a beam with a uniform intensity profile. For a given tumor, the uniformity of hyperthermia temperatures was found to be a function of frequency, with 4.75 MHz providing reasonably uniform results for typical tumor heights (near 7 mm). For ablation, diffraction computations were employed to calculate beam intensity profiles; results show an initially rapid rise in temperature levels with subsequent, slower heating beyond the -3-dB limits of the focal volume. The model is now being refined, and additional phenomena, including nonlinear propagation, will be incorporated.

Ultrasonically induced disruption and hemolysis of vitreous hemorrhages.

High-intensity focused ultrasound was used to insonify simulated vitreous hemorrhages in two in vitro models and one in vivo animal model. Histopathologic evaluation of the treatment effect was performed with light and electron microscopy. Ultrasonic parameters resulting in histopathologically proven disruption and hemolysis of intravitreal blood were investigated. Most important in obtaining this effect were the temporal average acoustic power and pulse repetition rate of the applied ultrasonic beam. Accurate positioning of the therapeutic beam and visualization of internal spatio-temporal response patterns in the blood during insonification were monitored via simultaneous diagnostic B-mode imaging. Results suggested that high-intensity focused ultrasound might facilitate the two major mechanisms involved in the natural resorption process of vitreous hemorrhages namely hemolysis and surface-dependent phagocytosis.

Ultrasonic tissue characterization and histopathology in tumor xenografts following ultrasonically induced hyperthermia.

Cells derived from human skin malignant melanoma were implanted subcutaneously in athymic nude mice. Tumors which developed at the implant site were treated with ultrasonically induced hyperthermia at 49 degrees C for 30 min. Tumors were scanned with a computerized diagnostic ultrasound system before and after treatment. Light (LM) and electron (EM) micrographs of tumors were obtained after scanning. Changes in ultrasonic tissue characterization parameters following treatment were well correlated with histopathologic changes observed in tumors. The results are significant in terms of clinical application of ultrasonically induced hyperthermia for treatment of intraocular tumors and the noninvasive monitoring of tumors by use of diagnostic ultrasound.

Therapeutic ultrasound.

Currently the most widely applied use of therapeutic ultrasound in ophthalmology is for the treatment of refractory glaucoma. This new technique is described, histologic changes in experimental animals discussed, and mode of action postulated. The intraocular pressure (IOP) results obtained after treatment of 187 patient eyes are presented, with a review of complications and visual acuity changes. This study demonstrates that 70% of patients treated had an IOP reduction to 25 mm Hg or less one year after ultrasound therapy.

A small portable proton exchange membrane fuel cell and hydrogen generator for medical applications.

Small, lightweight power sources for total artificial hearts (TAH), left ventricular assist devices (LVAD), and other medical products are under development. The new power source will provide 2 to 3 times the capacity of conventional batteries. The implications of this new power source are profound. For example, for the Heartmate LVAD, 5 to 8 hours of operation are obtained with 3 lb of lead acid batteries (Personal Communication Mr. Craig Sherman, Thermo Cardiosystems, Inc TCI 11/29/96). With the same weight, as much as 14 hours of operation appear achievable with the proton exchange membrane (PEM) fuel cell power source. Energy densities near 135 watt-hour/L are achievable. These values significantly exceed those of most conventional and advanced primary and secondary batteries. The improvement is mission dependent and even applies for the short deployment cited above. The comparison to batteries becomes even more favorable if the mission length is increased. The higher capacity requires only replacement of lightweight hydride cartridges and logistically available water. Therefore, when one spare 50 L hydride cartridge weighing 115 g is added to the reactant supply the energy density of the total system increases to 230 watt-hour/kg. This new power source is comprised of a hydrogen fueled, air-breathing PEM fuel cell and a miniature hydrogen generator (US Patent No 5,514,353). The fuel cell is of novel construction and differs from conventional bipolar PEM fuel cells by the arrangement of cells on a single sheet of ion-exchange membrane. The construction avoids the weight and volume penalty of conventional bipolar stacks. The hydrogen consumed by the fuel cell is generated load-responsively in the miniature hydrogen generator, by reacting calcium hydride with water, forming in the process hydrogen and lime. The generator is cartridge rechargeable and available in capacities providing up to several hundred watt-hours of electric power.

Diagnostic ultrasound in dermatology: current uses and future potential.

Although ultrasound has been used for many years for a variety of medical diagnostic purposes, only recently have systems been designed that allow for its application to skin. High-resolution ultrasound systems now permit accurate, quantitative, noninvasive assessment of skin and cutaneous diseases. This technique will assist in the management of both inflammatory and neoplastic processes. Scans of skin obtained with a prototype high-resolution ultrasound system are presented. The computer-assisted creation of three-dimensional images from sequential B-mode scans is described.