Use of gene therapy to suppress the antigen-specific immune responses in mice to an HLA antigen.

Hematopoietic chimerism has been used in the laboratory to induce life-long immunologic tolerance to donor antigens. The present study demonstrates that mice transplanted with autologous bone marrow cells retrovirally transduced to express HLA-A2.1 develop a significantly depressed immune response to this antigen while retaining normal reactivity to HLA-B7. Retrovirus-mediated transduction was performed using whole bone marrow-producer cell coculture. This approach did not result in significant gene transfer into hematopoietic progenitor cells. Despite this, the antibody response to HLA-A2.1 in mice reconstituted with genetically modified BMC was completely suppressed three months following bone marrow transplantation. Cell-mediated immunity to HLA-A2.1 was partially suppressed in three-fourths of animals tested three months later, although one animal had a CTL profile similar to that an of HLA-A2.1 transgenic mouse. Complete suppression of the antibody-mediated immune response occurred when only one-third of mice had evidence of the introduced genes in their spleen and one-tenth had the introduced sequences in their circulating WBCs by PCR. In conclusion, engineering of BMC to express donor MHC genes may be an alternative to xenogeneic BMT to induce chimerism and tolerance. More efficient transduction of bone marrow progenitor cells may result in more persistent gene expression and long-lasting transplantation tolerance in recipients of genetically modified bone marrow. Successful application of this technology may also be useful in altering immune responses to other external and self antigens.

Extracorporeal liver perfusion using human and pig livers for acute liver failure.

BACKGROUND: Patients with fulminant hepatic failure (FHF) often die awaiting liver transplantation. Extracorporeal liver perfusion (ECLP) has been proposed as a method of "bridging" such patients to transplantation. We report the largest experience to date of ECLP using human and porcine livers in patients with acute liver failure. METHODS: Patients with FHF unlikely to survive without liver transplantation were identified. ECLP was performed with human or porcine livers. Patients underwent continuous perfusion until liver transplantation or withdrawal of support. Two perfusion circuits were used: direct perfusion of patient blood through the extracorporeal liver and indirect perfusion with a plasma filter between the patient and the liver. FINDINGS: Fourteen patients were treated with 16 livers in 18 perfusion circuits. Nine patients were successfully "bridged" to transplantation. ECLP stabilized intracranial pressure (ICP) and cerebral perfusion pressure (CPP). Arterial ammonia levels fell from a median of 146 to 83 micromol/liter within 12 hr and this reduction was maintained at least 48 hr. Pig and human ECLP lowered ammonia levels equally. Serum bilirubin levels also fell from a median of 385 to 198 micromol/liter over the first 12 hr but the response was not sustained as well with porcine livers. There was no immunological benefit to using the the filtered perfusion circuit. INTERPRETATION: These data demonstrate that ECLP is safe and can provide metabolic support for comatose patients with fulminant hepatic failure for up to 5 days. While labor and resource intensive, this technology is available to centers caring for patients with acute liver failure and deserves wider evaluation and application.

Treatment of carbon tetrachloride and phenobarbital-induced chronic liver failure with intrasplenic hepatocyte transplantation.

Hepatocyte transplantation (HTx) has been shown to improve the survival of laboratory animals with experimentally induced acute liver failure and to ameliorate the physiologic abnormalities associated with liver-based metabolic deficiencies. However, the role of HTx in the treatment of liver cirrhosis (LC) has not been adequately studied. In order to address this issue, HTx was performed in rats following induction of stable LC using phenobarbital (PhB) and carbon tetrachloride (CCl4). Intrasplenic transplantation of 50 x 10(6) primary hepatocytes could significantly improve liver functions and prolong the survival of rats with irreversible, decompensated LC.

The effect of drying, heat, and pH on the survival of Legionella pneumophila.

The susceptibility of Legionella pneumophila, Philadelphia 1 strain, to drying, pH variation, and heating was determined and compared to that of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. L. pneumophila was quite sensitive to drying, with a four log drop in viability occurring during the first 30 seconds. After 90 minutes of drying in tap water, some samples contained no viable organisms, whereas E. coli, S. aureus, and P. aeruginosa still contained several thousand viable organisms. L. pneumophila showed a two log drop in viable cells after being held for one month in tap water varying in pH from 4 to 7. This was similar to what was found for P. aeruginosa. However, at pH 8 there was a six log drop in viability for L. pneumophila which was not evident with P. aeruginosa. At pH 2 to 3, both organisms could only survive short term (minutes) exposure. Heating experiments revealed a four log drop in viability for L. pneumophila at temperatures ranging from 55 to 65 degrees C. The survival of E. coli and S. aureus was greater at these temperatures.

The development of technology competencies and training guidelines for occupational therapists.

The ability to use technology has become a survival skill in our society. This paper discusses technology trends related to the demands of the Information Age, the increasing availability of information and assistive technologies, and the impact of recent civil rights legislation mandating that persons with disabilities be given equal access to technologies that can enhance functional performance. Occupational therapists must become competent in the application and integration of these technologies into reasonable accommodation interventions if we are to meet the changing needs of persons with disabilities. To address this need for technology training, a multitiered set of technology competencies specifically designed for occupational therapy practitioners was authored by the American Occupational Therapy Association Technology Special Interest Section and reviewed by occupational therapists with technology expertise. The process of developing these competencies and recommendations for implementing them within occupational therapy educational programs are discussed.

The Vocational Training FacilityAn Interactive Learning Program to Return Persons With Physical Disabilities to Employment.

This paper describes the results of the program-development phase of the Vocational Training Facility (VTF) taking place at the Palo Alto Veterans Affairs Medical Center Rehabilitation Research and Development Center. The VTF staff has developed a self-paced, multimedia curriculum comprised of adapted training packages, interactive videos, and additional training and testing materials designed to teach entry-level desktop publishing and reasonable accommodation skills to individuals with spinal cord injuries. The curriculum is taught via the Macintosh™ computer to allow independent, "hands-off" access to training materials. Each student is given an integrated workstation that is equipped with the Desktop Vocational Assistant Robot (De VAR); a set of low-and high-technology assistive hardware, software, and devices; and ergonomic furniture and adaptations customized to fit individual learning and access needs. Each student completes a 12-week, full-time training program followed by a 3-month internship with a local corporate sponsor. This paper summarizes the evaluation results of the VTF program by the first nine students, with spinal cord injuries ranging paraplegia to high-level quadriplegia, who have completed the program.

Stimulation of proteinase biosynthesis by canavanine in streptococcus faecalis var. liquefaciens.

l-Canavanine, an analogue of arginine, was found to stimulate the synthesis of an extracellular proteinase in Streptococcus faecalis var. liquefaciens. Cells grown in a synthetic medium containing 10(-4)m arginine and 10(-4)m canavanine produced almost twice as much proteinase as cells grown in 2 x 10(-3)m arginine alone; total growth was the same in both media. Hydrolyzed proteinase samples were analyzed for arginine and canavanine by means of paper chromatography and electrophoresis. Arginine, but not canavanine, was detected in the purified enzyme sample.

Regulation of extracellular protease formation by Serratia marcescens.

Heavy cell suspensions of Serratia marcescens, when grown in gelatin-containing media, produce extracellular proteases which increase in specific activity in a linear fashion for 3 to 4h. During partial purification, a single peak of proteolytic activity was demonstrated by Sephadex G-100 chromatography. However, electrophoresis using 5% polyacrylamide gels discloses three proteolytically active bands. Evidence in favor of gelatin acting as an inducer of the 'proteolytic system' was provided by two observations. First, proteolytic activity is only present in media containing gelatin. Secondly, the addition of 10(-4) M rifampicin to cells growing in gelatin-containing medium plus an additional carbon source inhibits protease activity totally, but has no effect on growth. When glycerol is added to a growing cell suspension in gelatin-containing medium, growth increases, but protease specific activity decreases. This 'glycerol effect' is not due to an accumulation of active or inactive enzyme in association with the cell, nor to a decrease in the total number of proteases synthesized. Rather, glycerol, as other utilizable carbohydrates, exerts a repression which can be eliminated by 5 mM dibutyryl cyclic AMP.

Staphylococcal bacteriophage-associated lysin: a lytic agent active against Staphylococcus aureus.

A lytic enzyme active against viable, intact staphylococci is released into culture fluids upon lysis of bacteriophage-infected Staphylococcus aureus PS53 cells. This enzyme, staphylococcal phage-associated lysin (PAL), was partially purified by ammonium sulfate precipitation and gel filtration through Sephadex G-200. PAL is optimally active at pH 6.5 and 30 C, and lytic activity is greatly enhanced by the addition of reducing agents. Lytic activity was observed against all strains of staphylococci tested and against purified staphylococcal cell walls, but no activity was noted against other bacterial species. PAL possesses peptidase activity and results in the production of spheroplasts which can be osmotically stabilized for extended periods by the addition of 7.5% polyethylene glycol 4000.

A self-limited febrile illness produced in guinea pigs associated with oral administration of Legionella pneumophila.

The presumed route of human infection by Legionella pneumophila is inhalation. We investigated possible oral transmission of legionellosis in guinea pigs. Fifty-six guinea pigs (group 1) were given virulent L. pneumophila, serogroup 1, in drinking water. Fifty-nine guinea pigs (group 2) were inoculated with L. pneumophila via gastric intubation. Nineteen guinea pigs (group 3) were given heat-killed L. pneumophila in drinking water. Twenty-four guinea pigs (group 4, positive control) were inoculated intraperitoneally with L. pneumophila. Twenty-seven guinea pigs (group 5, negative control) were either intubated gastrically with phosphate-buffered saline or given drinking water without L. pneumophila. Sixty-six of 115 (57%) of the guinea pigs orally inoculated with viable L. pneumophila (groups 1 and 2) had a temperature greater than or equal to 103 degrees F and 8 of 115 (7%) had diarrhea, compared with 0 of 19 (0%) and 0 of 19 (0%), respectively, in group 3 and 1 of 27 (4%) and 0 of 27 (0%), respectively, in group 5. There were no fatalities in groups 1, 2, 3, and 5 compared with 15 of 24 (63%) in group 4. Groups 1, 2, and 4 consistently showed pneumonitis and splenitis. The pneumonitis of groups 1 and 2 was mild, predominantly interstitial, and mainly composed of macrophages; neither gross nor microscopic evidence of aspiration was seen. In group 1, 4 of 29 (14%) guinea pigs tested seroconverted to L. pneumophila compared with 0 of 7 (0%) in group 3 and 0 of 10 (0%) in group 5. In groups 1 and 2 combined, L. pneumophila was isolated from the lung of 5 of 57 (11%) guinea pigs and spleen of 5 of 47 (11%) guinea pigs compared with 0 of 14 guinea pigs in group 5. We conclude that viable L. pneumophila administered orally produces a self-limited febrile illness in guinea pigs.

Evaluation of a vocational robot with a quadriplegic employee.

A vocational robotic workstation capable of performing activities of daily living (ADL) and vocational tasks was placed for 18 months in the work site of an employee with C4 to C5 quadriplegia. A single-subject study was conducted to evaluate the performance of the robot vs that of a human attendant. The employee preferred the robot over the attendant for performance of all vocational tasks and ADLs, with the exception of feeding. Results indicated that the robot was capable of safely replacing the attendant for two five-hour periods during the workday, thus proving to be a cost-effective alternative to full-time, on-the-job attendant care. The study demonstrated the potential of robotics technology for returning independence and control to disabled employees and for offering corporate employers a solution to the problem of reasonable accommodation in the workplace.

Lack of airborne spread of infection by Legionella pneumophila among guinea pigs.

Many investigators find no spread of Legionnaires disease from person to person. The present study examined the question of airborne transmission of infection by Legionella pneumophila serogroup 1 from guinea pigs inoculated nasally with the agent to healthy guinea pigs. The nasal inoculation produced confluent peribronchiolar pneumonia similar to the pulmonary lesions observed in humans, but by techniques of clinical observation, serology, culture, and pathology, there was no evidence of airborne spread of infection from 26 inoculated guinea pigs to 64 uninoculated guinea pigs. The results, compatible with epidemiological studies of Legionnaires disease that fail to demonstrate airborne person-to-person transmission of the illness in humans, are useful for scientists who work with animal models of Legionnaires disease.

Pleomorphism of Legionella pneumophila.

Legionella pneumophila (Lp), serogroups 1-6, was grown in vitro on a variety of media, in embryonated hens' eggs, and in guinea pigs. The morphology of the microbe was examined by light, immunofluorescent, and electron microscopy (transmission, scanning, negative staining). The configuration of all serogroups examined differed somewhat on agar media, in liquid media, and in vivo. Each serogroup of Lp showed pleomorphic features indistinguishable from the others. Except for filamentous forms, pleomorphism was least conspicuous on agar. By contrast, pleomorphism was most apparent in yeast extract broth, and it was detected by all of the morphologic techniques employed. Bacilli were seen most commonly, but the spectrum of forms was as follows: cocci, coccobacilli (short bacilli), medium bacilli, bacilli with terminal cocci, filamentous forms, and branches. Diplococci, branches, and stalks were only rarely seen, and the latter form was never visualized by immunofluorescence. In tissue samples from infected guinea pigs and embryonated hens' eggs, Lp was typically a short bacillus, but coccoid and coccobacillary forms were seen. Lp is clearly a pleomorphic bacterium, particularly when grown in yeast extract broth. The variety of forms described herein might provide clues to taxonomy, ecologic niche, and physiology of Lp.

Effect of interleukin-1 alpha on the in vitro activation of tumor-draining lymph node cells for adoptive immunotherapy.

Adoptive immunotherapy using in vitro activated T-cells can mediate the destruction of metastatic tumor deposits in animal models. These antitumor effector cells can be generated from mice with subcutaneous tumor by the sequential in vitro activation of tumor-draining lymph node (TDLN) cells with monoclonal antibody to the T-cell receptor complex (anti-CD3) and expansion in low concentrations of interleukin-2 (IL-2). In this animal model, the concomitant presence of visceral tumor can suppress the sensitization of tumor-reactive TDLN cells. We investigated whether IL-1 alpha added during in vitro activation and/or expansion of TDLNs could augment their antitumor activity in adoptive therapy. Mice were inoculated subcutaneously with MCA 205 tumor. TDLN cells were harvested and activated in vitro with 1 microgram/ml anti-CD3 for 2 days (anti-CD3 phase), followed by expansion in 10 U/ml IL-2 for 3 days (IL-2 phase). Experimental cultures had IL-1 (10-10,000 U/ml) added in either or both phases. After the 5-day culture period, cells were counted to determine in vitro cellular proliferation and then adoptively transferred to mice bearing 3-day established lung metastases to assess in vivo antitumor efficacy. IL-1 added during the anti-CD3 or IL-2 phase did not alter in vitro cellular proliferation. The presence of IL-1 during the anti-CD3 phase led to the generation of cells that were significantly more therapeutically efficacious than cells generated in the absence of IL-1. The effect of IL-1 during anti-CD3 activation appeared to be dose dependent in the concentration range 10-1,000 U/ml. The addition of IL-1 during the IL-2 phase only did not enhance the antitumor reactivity of the activated cells. The beneficial effect of IL-1 during the anti-CD3 activation phase was specific for the tumor against which the TDLN had been initially sensitized; also, there was no evidence that non-tumor bearer lymphocytes developed significant antitumor reactivity when "activated" with IL-1 and anti-CD3. Furthermore, addition of IL-1 during the anti-CD3 activation phase abrogated suppression induced by the concomitant presence of lung metastases during subcutaneous tumor growth. IL-1 appears to up-regulate the in vitro activation of tumor-reactive lymphocytes derived from TDLN, in both the presence and the absence of visceral metastases.

Hepatocyte transplantation in rats with decompensated cirrhosis.

Hepatocyte transplantation improves the survival of laboratory animals with experimentally induced acute liver failure and the physiological abnormalities associated with liver-based metabolic deficiencies. The role of hepatocyte transplantation in treating decompensated liver cirrhosis, however, has not been studied in depth. To address this issue, cirrhosis was induced using phenobarbital and carbon tetrachloride (CCL(4)) and animals were studied only when evidence of liver failure did not improve when CCL(4) was held for 4 weeks. Animals received intrasplenic transplantation of syngeneic rat hepatocytes (G1); intraperitoneal transplantation of syngeneic rat hepatocytes (G2); intraperitoneal transplantation of a cellular homogenate of syngeneic rat hepatocytes (G3); intraperitoneal transplantation of syngeneic rat bone marrow cells (G4); or intrasplenic injection of Dulbecco's modified Eagle medium (DMEM) (G5). After transplantation, body weight and serum albumin levels deteriorated over time in all control (G2-G5) animals but did not deteriorate in animals receiving intrasplenic hepatocyte transplantation (G1) (P <.01). Prothrombin time (PT), total bilirubin, serum ammonia, and hepatic encephalopathy score were also significantly improved toward normal in animals receiving intrasplenic hepatocyte transplantation (P <. 01). More importantly, survival was prolonged after a single infusion of hepatocytes and a second infusion prolonged survival from 15 to 128 days (P <.01). Thus, hepatocyte transplantation can improve liver function and prolong the survival of rats with irreversible, decompensated cirrhosis and may be useful in the treatment of cirrhosis in humans.