A peptide derived from the intercellular adhesion molecule-2 regulates the avidity of the leukocyte integrins CD11b/CD18 and CD11c/CD18.

beta 2 integrin (CD11a,b,c/CD18)-mediated cell adhesion is required for many leukocyte functions. Under normal circumstances, the integrins are nonadhesive, and become adhesive for their cell surface ligands, the intercellular adhesion molecules (ICAMs), or soluble ligands such as fibrinogen and iC3b, when leukocytes are activated. Recently, we defined a peptide derived from ICAM-2, which specifically binds to purified CD11a/CD18. Furthermore, this peptide strongly induces T cell aggregation mainly mediated by CD11a/CD18-ICAM-1 interaction, and natural killer cell cytotoxicity. In the present study, we show that the same ICAM-2 peptide also avidly binds to purified CD11b/CD18, but not to CD11c/CD18. This binding can be blocked by the CD11b antibody OKM10. The peptide strongly stimulates CD11b/CD18-ICAM-1-mediated cell aggregations of the monocytic cell lines THP-1 and U937. The aggregations are energy and divalent cation-dependent. The ICAM-2 peptide also induces CD11b/CD18 and CD11c/CD18-mediated binding of THP-1 cells to fibrinogen and iC3b coated on plastic. These findings indicate that in addition to induction of CD11a/CD18-mediated cell adhesion, the ICAM-2 peptide may also serve as a "trigger" for high avidity ligand binding of other beta 2 integrins.

Tumor targeting with a selective gelatinase inhibitor.

Several lines of evidence suggest that tumor growth, angiogenesis, and metastasis are dependent on matrix metalloproteinase (MMP) activity. However, the lack of inhibitors specific for the type IV collagenase/gelatinase family of MMPs has thus far prevented the selective targeting of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) for therapeutic intervention in cancer. Here, we describe the isolation of specific gelatinase inhibitors from phage display peptide libraries. We show that cyclic peptides containing the sequence HWGF are potent and selective inhibitors of MMP-2 and MMP-9 but not of several other MMP family members. Our prototype synthetic peptide, CTTHWGFTLC, inhibits the migration of human endothelial cells and tumor cells. Moreover, it prevents tumor growth and invasion in animal models and improves survival of mice bearing human tumors. Finally, we show that CTTHWGFTLC-displaying phage specifically target angiogenic blood vessels in vivo. Selective gelatinase inhibitors may prove useful in tumor targeting and anticancer therapies.

Methylxanthine-evoked perturbation of spontaneous and evoked activities in isolated newborn rat hippocampal networks.

Treatment of apnea of prematurity with methylxanthines like caffeine, aminophylline or theophylline can evoke hippocampal seizures. However, it is unknown at which interstitial brain concentrations methylxanthines promote such neonatal seizures or interfere with physiological 'early network oscillations' (ENOs) that are considered as pivotal for maturation of hippocampal neural networks. We studied theophylline and caffeine effects on ENOs in CA3 neurons (CA3-ENOs) and CA3 electrical stimulation-evoked monosynaptic CA1 field potentials (CA1-FPs) in sliced and intact hippocampi, respectively, from 8 to 10-days-old rats. Submillimolar doses of theophylline and caffeine, blocking adenosine receptors and phosphodiesterase-4 (PDE4), did not affect CA3-ENOs, ENO-associated cytosolic Ca(2+) transients or CA1-FPs nor did they provoke seizure-like discharges. Low millimolar doses of theophylline (⩾1mM) or caffeine (⩾5mM), blocking GABAA and glycine receptors plus sarcoplasmic-endoplasmic reticulum Ca(2+) ATPase (SERCA)-type Ca(2+) ATPases, evoked seizure-like discharges with no indication of cytosolic Ca(2+) dysregulation. Inhibiting PDE4 with rolipram or glycine receptors with strychnine had no effect on CA3-ENOs and did not occlude seizure-like events as tested with theophylline. GABAA receptor blockade induced seizure-like discharges and occluded theophylline-evoked seizure-like discharges in the slices, but not in the intact hippocampi. In summary, submillimolar methylxanthine concentrations do not acutely affect spontaneous CA3-ENOs or electrically evoked synaptic activities and low millimolar doses are needed to evoke seizure-like discharges in isolated developing hippocampal neural networks. We conclude that mechanisms of methylxanthine-related seizure-like discharges do not involve SERCA inhibition-related neuronal Ca(2+) dysregulation, PDE4 blockade or adenosine and glycine receptor inhibition, whereas GABA(A) receptor blockade may contribute partially.

Expression and characterization of a B cell growth promoting polypeptide derived from the 12 kDa B cell growth factor gene (BCGF 1).

The expression and partial purification of recombinant 12 kDa B cell growth factor are reported. The polypeptide was derived from the genomic sequence of the gene (BCGF 1) which is here shown to be a single copy gene that localizes to human chromosome 16. When expressed as a glutathione S-transferase fusion protein in E. coli, the protein appears as a 38 kDa polypeptide in Western blot analysis using a peptide antibody. The purified fusion protein stimulates the proliferation of activated human B cells in a dose-dependent manner, and the active site resides within the 104 carboxy-terminal amino acids. The availability of biologically active recombinant 12 kDa B cell growth factor will enable its evaluation in B cell growth regulation, and provides a new means of in vitro culturing of human B lymphocytes.

Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2.

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.

Leukocyte adhesion--an integrated molecular process at the leukocyte plasma membrane.

Leukocyte adhesion is of pivotal functional importance, because most leukocyte functions depend on cell-cell contact. It must be strictly controlled, both at the level of specificity and strength of interaction, and therefore several molecular systems are involved. The most important leukocyte adhesion molecules are the selectins, the leukocyte-specific beta2-integrins and the intercellular adhesion molecules. The selectins induce an initial weak contact between cells, whereas firm adhesion is achieved through integrin intercellular adhesion molecular binding. Although studies during the past twenty years have revealed several important features of leukocyte adhesion much is still poorly understood, and further work dealing with several aspects of adhesion is urgently needed. In this short essay, we review some recent developments in the field.

Leukocyte adhesion--a fundamental process in leukocyte physiology.

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte beta 2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.

Perspectives on the role of afferent signals in control of motor neuroprostheses.

K.O. Johnson reviews the architecture and low level neural mechanisms by which the external environment is transduced and encoded into the neural system, summarizing work that correlates neurophysiological and psychophysical testing with isolation of sensory components. The slowly adapting Type I afferent system is responsible for form and texture perception; the rapidly adapting afferent system is responsible for motion perception; and the Pacinian corpuscle system is responsible for vibratory sensation. R.R. Riso reviews the current level of understanding of the major factors to be considered in the design of a functional neuromuscular stimulation (FNS) grasp controller that uses cutaneous sensory feedback to detect slip. The elegant natural control scheme that matches the ratio of grip and lift forces to frictional conditions provides a model for implementing a slip-based control algorithm. D. Popovic discusses the possible use of recordings from more proximal peripheral nerves to determine needed information for synthesis of locomotion. The discussion is illustrated with an animal model where rule-based closed-loop control is used for the ankle joint during treadmill locomotion. Neural signals from the tibial and superficial peroneal nerves were employed to substitute for missing afferent input from cutaneous and proprioceptive sensors. The feasibility of more invasive intraneural electrodes for distinguishing sensory from motor information in mixed nerves is considered. M. Koris raises surgical and functional issues relevant to developing clinical FNS systems. C. Van Doren suggests alternative neurophysiological and engineering approaches.

Electrical stimulation to restore respiration.

Electrical stimulation has been used for over 25 years to restore breathing to patients with high quadriplegia causing respiratory paralysis and patients with central alveolar hypoventilation. Three groups have developed electrical pacing systems for long-term support of respiration in humans. These systems consist of electrodes implanted on the phrenic nerves, connected by leads to a stimulator implanted under the skin, and powered and controlled from a battery-powered transmitter outside the body. The systems differ principally in the electrode design and stimulation waveform. Approximately 1,000 people worldwide have received one of the three phrenic pacing devices, most with strongly positive results: reduced risk of tracheal problems and chronic infection, the ability to speak and smell more normally, reduced risk of accidental interruption of respiration, greater independence, and reduced costs and time for ventilatory care. For patients with partial lesions of the phrenic nerves, intercostal muscle stimulation may supplement respiration.

Challenges to clinical deployment of upper limb neuroprostheses.

The technology for functional neuromuscular stimulation (FNS) as a means of providing upper limb function to people with tetraplegia has been under development by three clinical research groups for almost two decades. This paper presents the current status of the clinical trials of three FNS systems: a noninvasive system built into a cosmetic forearm splint, a 30-channel percutaneous system, and an 8-channel implantable system. The complexity of FNS systems and the unique characteristics of the individuals to whom they are applied combine to create many clinical and technical challenges that must be addressed before the devices can be deployed. The emerging challenges to widespread clinical introduction of FNS systems for hand and arm function are identified and analyzed. In addition to the demands of designed and conducting the clinical trials to satisfy regulatory requirements, the lack of knowledge, skepticism, and the complacency on the part of potential FNS recipients, as well as of rehabilitation professionals, must be overcome through education and careful consideration of economic and societal factors in the design of clinical systems.

Dynamic myoplasty: surgical transfer and stimulation of skeletal muscle for functional substitution or enhancement.

Dynamic myoplasty combines muscle transfer with electrical stimulation to provide contractile function that augments or replaces impaired organ function. Dynamic cardiomyoplasty was the first clinical application in which a skeletal muscle, latissimus dorsi, was transferred and stimulated to provide cardiac assistance, a function different from its original one. The problem of early muscle fatigue that was encountered in the initial implementation of the method was solved by training the muscle with electrical stimulation and thus changing its fiber composition. With intramuscular electrodes, the conditioned latissimus dorsi is stimulated in synchrony with the heart muscle. Safeguards are built into the two-channel implanted stimulator to avoid excessively high pulse rates. Clinicians report that 80% of patients with moderate to severe heart failure prior to operation showed a clinical improvement of 1.6 New York Heart Association classes. Alternative methods of providing cardiac assistance that are also being investigated include wrapping the muscle around the aorta, creating a skeletal muscle ventricle, and using the muscle to power an implantable pump. These latter techniques are still under preclinical investigation. Compared with heart transplant, cardiomyoplasty has the great advantage of not being subject to tissue rejection. The second principal application of dynamic myoplasty is treatment of fecal incontinence through creation of an electrically stimulated skeletal muscle neosphincter (ESMNS). The gracilis muscle of the leg is mobilized, wrapped around the anal canal, and conditioned with electrical stimulation to become more fatigue resistant. To achieve continence, the muscle is continuously stimulated except when the patient wishes to defecate. Overall success rates in achieving continence are 60-65%. Both cardiomyoplasty and the ESMNS technique, and their associated devices, are being refined through ongoing clinical trials.

New control strategies for neuroprosthetic systems.

The availability of techniques to artificially excite paralyzed muscles opens enormous potential for restoring both upper and lower extremity movements with neuroprostheses. Neuroprostheses must stimulate muscle, and control and regulate the artificial movements produced. Control methods to accomplish these tasks include feedforward (open-loop), feedback, and adaptive control. Feedforward control requires a great deal of information about the biomechanical behavior of the limb. For the upper extremity, an artificial motor program was developed to provide such movement program input to a neuroprosthesis. In lower extremity control, one group achieved their best results by attempting to meet naturally perceived gait objectives rather than to follow an exact joint angle trajectory. Adaptive feedforward control, as implemented in the cycle-to-cycle controller, gave good compensation for the gradual decrease in performance observed with open-loop control. A neural network controller was able to control its system to customize stimulation parameters in order to generate a desired output trajectory in a given individual and to maintain tracking performance in the presence of muscle fatigue. The authors believe that practical FNS control systems must exhibit many of these features of neurophysiological systems.

Technology transfer of neuroprosthetic devices.

Despite long development periods for neuroprosthetic devices, the numbers in clinical use or clinical trials are rising, with an estimated 3,000 systems in use today. As they gain experience with the regulatory approval process, developers are learning to conduct research to best prepare for transfer of technology to industry. The track record of the first motor prosthesis to be approved by the United States Food and Drug Administration contains important lessons for a company planning to undergo the regulatory process. Throughout the development of a neuroprosthesis, the capabilities and preferences of the customers who will use it (physicians, surgeons, therapists, and end-users) should be sought out and used in device design. When a device has reached clinical application, particular attention is needed to maximize both the population who will use it and each individual's degree of use (optimal, partial, reluctant). Identification of person-technology mismatches can help to select training strategies and other interventions that can be applied to ensure a good rehabilitation outcome.

Neural signals for command control and feedback in functional neuromuscular stimulation: a review.

In current functional neuromuscular stimulation systems (FNS), control and feedback signals are usually provided by external sensors and switches, which pose problems such as donning and calibration time, cosmesis, and mechanical vulnerability. Artificial sensors are difficult to build and are insufficiently biocompatible and reliable for implantation. With the advent of methods for electrical interfacing with nerves and muscles, natural sensors are being considered as an alternative source of feedback and command signals for FNS. Decision making methods for higher level control can perform equally well with natural or artificial sensors. Recording nerve cuff electrodes have been developed and tested in animals and demonstrated to be feasible in humans for control of dorsiflexion in foot-drop and grasp in quadriplegia. Electromyographic signals, being one thousand times larger than electroneurograms, are easier to measure but have not been able to provide reliable indicators (e.g., of muscle fatigue) that would be useful in FNS systems. Animal studies have shown that information about the shape and movement of arm trajectories can be extracted from brain cortical activity, suggesting that FNS may ultimately be directly controllable from the central nervous system.

A leukocyte integrin binding peptide from intercellular adhesion molecule-2 stimulates T cell adhesion and natural killer cell activity.

Adhesion is of pivotal importance for a number of leukocyte functions. Little is known about the binding between leukocyte integrins and the intercellular adhesion molecules (ICAMs). Normally integrins are nonadhesive, and require a stimulus to become active. We have now identified a peptide from ICAM-2, which binds to leukocyte integrins and activates adhesion. Furthermore, the peptide strongly increased the binding and cytotoxicity of natural killer cells. These findings show that adhesion-dependent leukocyte functions can be activated by ligand-derived peptides, and therefore provide evidence that the avidity of leukocyte integrins is up-regulated by integrin-ligand interactions.

Immunological mapping of the human leucocyte adhesion glycoprotein gp90 (CD18) by monoclonal antibodies.

The leucocyte surface glycoproteins CD11a (gp160, LFA-1 antigen, TA-1 antigen), CD11b (gp155, Mac-1 antigen, OKM1 antigen, Mo-1 antigen), CD11c (gp130, Leu-M5 antigen), and CD18 (gp90) constitute three heterodimers with different alpha chains and a common beta chain. Monoclonal antibodies to CD11a, b, or c block adhesion of certain types of leucocytes only, while several antibodies to CD18 inhibit adhesion in all of them. The functionally important site or sites on CD18 are not known. We have now isolated the CD11a,b,c-CD18 leucocyte antigen complex in large amounts from human leucocytes, and produced several new monoclonal antibodies reacting with CD18. One of these antibodies, like those described earlier, inhibits leucocyte adhesion, whereas the others do not. By means of competition experiments, at least four epitope regions were found. These antibodies should be valuable in elucidating the regions essential in CD18-mediated leucocyte functions.

Structural study of the sugar chains of human leukocyte cell adhesion molecules CD11/CD18.

Leu-CAMs (CD11/CD18) consisting of LFA-1, Mac-1, and p150/95 are leukocyte cell surface glycoproteins that are involved in various leukocyte functions. The asparagine-linked sugar chains were released as oligosaccharides from Leu-CAMs by hydrazinolysis. About 12 mol of sugar chains was released from 1 mol of Leu-CAMs. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. The neutral and sialdase-treated acidic oligosaccharides were fractionated by chromatography on lectin columns followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that Leu-CAMs contain mainly high mannose type and high molecular weight complex type sugar chains. The latter sugar chains were of bi-, tri-, and tetraantennary complex types with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and/or the Gal beta 1----3GlcNAc beta 1----groups together with the Gal beta 1----4GlcNAc group in their outer-chain moieties. In addition to these sugar chains, a small amount of monoantennary complex type and hybrid type sugar chains was found in Leu-CAMs. Furthermore, analysis of the asparagine-linked sugar chains released from the beta-subunit of Leu-CAMs by a series of lectin chromatography showed that subunit-specific glycosylation is not observed between the alpha- and beta-subunits of Leu-CAMs.