Adipose tissue: a new target for electroporation-enhanced DNA vaccines.

DNA vaccines delivered using electroporation (EP) have had clinical success, but these EP methods generally utilize invasive needle electrodes. Here, we demonstrate the delivery and immunogenicity of a DNA vaccine into subcutaneous adipose tissue cells using noninvasive EP. Using finite element analysis, we predicted that plate electrodes, when oriented properly, could effectively concentrate the electric field within adipose tissue. In practice, these electrodes generated widespread gene expression persisting for at least 60 days in vivo within interscapular subcutaneous fat pads of guinea pigs. We then applied this adipose-EP protocol to deliver a DNA vaccine coding for an influenza antigen into guinea pigs. The resulting host immune responses elicited were of a similar magnitude to those achieved by skin delivery with EP. The onset of the humoral immune response was more rapid when the DNA dose was spread over multiple injection sites, and increasing the voltage of the EP device increased the magnitude of the immune response. This study supports further development of EP protocols delivering gene-based therapies to subcutaneous fat.

Reduction of phagosomes in the vitiligo (C57BL/6-mivit/mivit) mouse model of retinal degeneration.

PURPOSE: The vitiligo (C57BL/6-mivit/mivit) mouse has a slowly progressing retinal degeneration, in which photoreceptor cell nuclei are gradually lost and the retinal pigment epithelium (RPE) is unevenly pigmented. The purpose of the present study was to assess the phagocytic ability of the RPE in the vitiligo mouse by determining whether and when a phagocytic burst occurs in affected mice and whether the number of phagosomes varies between control and affected animals. METHODS: Eyes of control and vitiligo mice 4 to 20 weeks of age were embedded in Spurr. Thin sections were cut and examined by electron microscopy to confirm the presence of phagosomes, particularly in the affected animals. Thick (1 micron) sections were cut, and quantitative morphometry was performed at the light microscope level. The length of RPE was determined, and phagosomes were counted in RPE cytoplasmic and microvillous areas. Data were expressed as phagosomes per 1000 microns. RESULTS: The vitiligo mouse has a peak phagocytic episode approximately 2 hours after light onset. The number of phagosomes in 4-week-old affected mice was significantly less than that in controls (13 phagosomes per 1000 microns compared to 30 phagosomes per 1000 microns). By week 8, the number was reduced to approximately 5 per 1000 microns. Phagosome number was not reduced further between weeks 8 and 20 in the affected animal. Macrophage-like cells containing pigment granules and phagosomes were observed in the subretinal space in areas where the rod outer segments had been separated from the RPE. CONCLUSIONS: The vitiligo mouse RPE contains phagosomes, but there are significantly fewer than in controls. It is not known whether a defect in RPE phagocytosis is the direct cause of the retinal defect in this model.

Effects of dark-rearing on the retinal degeneration of the C57BL/6-mivit/mivit mouse.

The C57BL/6J-mivit/mivit mouse has a retinal degeneration in which photoreceptor cells are lost slowly at a rate of about one row per month beginning at 8 weeks. ROS are severely disrupted by 4 months, but inner segments maintain a normal length through 6 months. In addition to photoreceptor changes, the retinal pigment epithelium is unevenly pigmented. The present study utilized histological and biochemical techniques to assess the effects of dark-rearing on the progression of the retinal degeneration in the mivit/mivit mouse at ages 4, 6, 8, 12, 16, 20, 24, and 28 weeks. Results of systematic morphometric evaluation indicated that the rate of loss of photoreceptor cells did not differ significantly from the rate determined for mivit/mivit animals reared in a standard light cycle. Furthermore, retinal detachment from RPE, the displacement of darkly-staining cells into the subretinal space and the influx of macrophage-like cells in the area of the ROS were still present in mivit/mivit animals reared in darkness. ROS of mivit/mivit seemed to be preserved for a slightly longer period of time in the dark-rearing condition. Rhodopsin levels in 4-week dark-reared mivit/mivit mice were 0.32 +/- 0.04 nmol per retina which was comparable to mivit/mivit mice reared under standard lighting conditions. At 20 and 28 weeks, rhodopsin levels decreased in mivit/mivit retinas to a similar level regardless of their lighting history. The findings of the study suggest that light deprivation does not retard the degeneration mivit/mivit retina. Results are discussed in comparison with effects of dark-rearing on other models of retinal degeneration.

Opsin synthesis in the C57BL/6-mivit/mivit mouse model of retinal degeneration.

The capacity of photoreceptor cells to synthesize opsin was evaluated in a newly-described mouse model of retinal degeneration, the C57BL/6-mivit/mivit. The mivit/mivit mouse loses photoreceptor cells at a rate of about one row per month beginning at 8 weeks, ROS are severely disrupted at 4 months, RPE is unevenly pigmented. Retinas of affected and control mice ages 4, 6, 8, 12, 16, 20, 24, 28, 32 and 52 weeks were incubated for 2 hours in medium containing [3H] leucine. Homogenates of retina samples were subjected to SDS-PAGE using disc gels. The gels were sliced and counted by scintillation. The incorporation of [3H] leucine into opsin was compared with its incorporation into other retinal proteins. During the early time points studied, mivit/mivit retinas incorporated proportionately similar amounts of [3H] leucine into opsin versus other retinal proteins as did controls. At 12 weeks, the percentage was about 80% and it continued to decline over the succeeding weeks studied. By 1 year, the proportion of leucine incorporated into opsin versus other proteins was only about 23% the amount incorporated in controls. The results of the present study suggest that the mivit/mivit photoreceptor cells are able to synthesize opsin and the gradual decline in synthetic ability follows the gradual loss of cells and is not correlated with the disruption of ROS.

Origin and propagation of elastogenesis in the developing cardiovascular system.

Ectomesenchyme derived from cardiac neural crest is critical to aorticopulmonary septation in the heart. However, any unique contribution of the cardiac ectomesenchyme to the extracellular matrix of the conotruncus has not been demonstrated previously. In this study the chronology and topography of soluble tropoelastin (STE) and the aldehyde-rich protein (ARP) of the elastic connective tissues have been examined in the chick embryo, stages 21-38, and in the quail-chick chimera, stages 24-35 (quail neural fold grafted onto a chick embryo). STE was located with immunofluorescence histochemistry, and ARP with Schiff's reagent. With these procedures prevenient sites of elastin synthesis are observed readily. The results show that the myocardium proper appears to have a role in the instigation of elastogenesis and in elastic fiber orientation; that the mesenchymal cells whose matrix contains elastic fibers are ectomesenchymal, of neural crest origin; and that elastin is deployed in an orderly proximal-distal sequence. It is hypothesized that elastogenesis is a critical event in aorticopulmonary septation.

A new interpretation of the direct Schiff reaction of elastic connective tissue.

A direct Schiff reaction of elastic tissues has been known for many years, but the nature of the native aldehyde-rich components has not been clear. In this study, chicken, quail, and rat embryos and adult rat lung, aorta, and kidney were fixed in methacarn or in a formalin solution, embedded in paraffin, and sections of 8-10 micron obtained. Rehydrated sections were incubated for various periods in solutions of the enzymes chondroitinase ABC, clostripain, collagenase, elastase, heparatinase, hyaluronidase, subtilisin Carlsberg ("protease"), or trypsin, and in solutions of phosphomolybdic acid or sodium borohydride. After incubation, sections were placed, without prior oxidation, in Schiff's reagent, and were ultimately observed and photographed in transmitted light or with blue or green epifluorescence. A Schiff-positive substance was found, always and exclusively, in elastic tissues of the vasculature and lungs, which was hydrolyzed by the proteolytic enzymes to an extent that ranged from complete loss of Schiff reaction in minutes (trypsin) to no loss of Schiff reaction in 22 hr (clostripain). The Schiff-reactive protein preceded the time of appearance of elastin in the early embryos. We conclude that the aldehyde-rich protein responsible for this reaction is a harbinger of elastogenesis in vivo and speculate that it may represent the elastic microfibril or a component thereof.

Toxicologic studies of a conjugate of asparaginase and polyethylene glycol in mice, rats, and dogs.

A conjugate of asparaginase and monomethoxypolyethylene glycol was evaluated in acute, subacute, and subchronic toxicologic studies in mice, rats, and dogs. The drug induced low-grade toxicosis. The appearance and behavior of rats and dogs were not affected by the treatment. Only large doses produced inactivity, loss of appetite, and loss of weight. The LD50 could not be established. The drug retarded slightly body weight gains in dogs and female rats and produced mild anemia in 30% of the female rats. Urinalysis and blood chemical determinations in rats and dogs were generally not affected by the treatment. Monomethoxypolyethylene glycol-asparaginase was detectable in the plasma of mice 13 days after IV, intraperitoneal, or IM administration, and in dogs for 3 to 4 weeks.

Iron exclusion from aflatoxin B1-induced hepatocellular carcinoma in rats.

Mixed-cell hepatocellular adenocarcinomas, induced by aflatoxin B1 (AFB1) in weanling rats, were allowed to develop for 18 months; age- and sex-matched controls were maintained. After 18 months, treated and control rats were administered iron dextran (ID), intraperitoneally (i.p.) as Nonemic. No gross or microscopic lesions were observed in control animals, while hepatic tumors were observed grossly in all AFB1-treated rats. Areas of apparently normal tissue, in histologic sections from control animals and in non-tumor sites of sections from AFB1-treated animals, were deeply stained for iron, both in endothelial cells and in hepatocytes, as visualized by Perls iron stain. Varying degrees of exclusion of stainable iron material were noted in histologic sections of lesions from AFB1-treated rats, as were alterations in endothelial cell staining characteristics. Iron staining clearly demonstrates the presence of varying cell populations within the induced mixed cell hepatocellular adenocarcinoma.

Cancer therapy with chemically modified enzymes. II. The therapeutic effectiveness of arginase, and arginase modified by the covalent attachment of polyethylene glycol, on the taper liver tumor and the L5178Y murine leukemia.

Monomethoxypolyethylene glycol (PEG) was attached covalently to arginase. PEG-arginase was effective in prolonging the survival times of mice injected with the Taper liver tumor, whereas unmodified arginase was ineffective. PEG-arginase was more effective than arginase in the in vitro destruction of L5178Y mouse leukemia. However, neither PEG-arginase nor arginase inhibited the in vivo growth of this tumor.

Metabolism of tRNA in rats with aflatoxin B1-induced hepatomas.

This study describes effects of aflatoxin B1-induced hepatomas on RNA metabolism in rats. At 4 and 24 hours after the administration of L-(14CH3)-methionine, tRNA was isolated from the livers and hydrolyzed enzymatically to nucleosides which were quantitatively measured by HPLC. Radioactivity of the nucleosides was also determined. The data indicate that although tRNA methylation may be more rapid in livers with hepatomas, catabolism of tRNA in tumorous tissue is slower than in control livers. The large increase in some radioactive methylated nucleosides and bases by the tumor-bearing rats during the 24-hour period following the administration of labeled methionine indicates increased turnover of mRNA and rRNA as well as tRNA. Since degradation of tumor tRNA appears to be delayed, the excessive amounts of the urinary methylated nucleosides must be derived from RNA in nonneoplastic tissue.

Comparison of urinary modified nucleosides and bases in rats with hepatomas and nephroblastomas.

Hepatomas were induced in rats with aflatoxin B1, and nephroblastomas with dimethylnitrosamine. Microscopic examination of livers of aflatoxin-treated rats revealed multinodular hepatocyte hyperplasia at 8 months, and by 13 months all rats had hepatomas. Nephroblastomas were observed by 4 months and by 8 months all rats had developed them. The urinary excretion of several modified nucleosides and bases by normal rats is dependent on body weight and reflects, to a certain extent, their concentrations in tissue tRNA. Increased levels of several modified nucleosides and bases were found in all rats that had cancer. Rats with hepatomas excreted essentially the same modified nucleosides and bases as did those with nephroblastomas; the quantitative patterns of excretion were different, however, suggesting that the urinary modified nucleosides and bases may be used to differentiate between neoplasms. Although the increase in urinary modified nucleosides and bases by tumor-bearing animals results primarily from more rapid turnover of neoplastic tRNAs, the data indicate that increased turnover of mRNA and possibly rRNA may occur in neoplastic tissue. Preliminary data suggest that increases in urinary modified nucleosides and bases may occur during a precancerous stage. The urinary pattern of modified nucleosides and bases by rats with hepatomas is altered if another primary tumor is present. The results obtained from these studies support the use of modified nucleosides and bases in urine as biochemical markers of cancer.

Clinical experience with the technetium-99 scan in children.

A study of 86 bone scans in 79 patients was conducted to determine ways to enhance the usefulness of this test and learn what factors influence its results. While the overall value of the bone scan as we initially used it was not what we had hoped, it could be improved substantially by utilizing all initial clinical information when ordering as well as interpreting the scan. Its main value in suspected sepsis is to localize affected areas in and around the pelvis and spine. The scan is often unnecessary in the diagnosis of long-bone osteomyelitis and is seldom indicated when diagnosing cellulitis or synovitis. In occult trauma it is valuable for further investigation of specific areas with possible nondisplaced fractures. Needle aspiration of the bone or joint does not seem to alter the scan. We conclude that the bone scan is chiefly valuable in locating focal areas of skeletal pathophysiology.

Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase.

An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.

Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.

Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.