Induction of primary cutaneous melanomas in C3H mice by combined treatment with ultraviolet radiation, ethanol and aloe emodin.

The role of ultraviolet (UV) radiation in the induction of nonmelanoma skin cancer is widely accepted, although its precise contribution to the development of primary cutaneous melanoma skin cancer requires further definition. We found that painting aloe emodin, a trihydroxyanthraquinone from Aloe barbadensis, in ethyl alcohol vehicle on the skin of mice in conjunction with exposure to UVB (280-320 nm) radiation results in the development of melanin-containing skin tumors. C3H/HeN mice were treated thrice weekly with aloe emodin in a 25% ethanol in water vehicle and exposed to 15 kJ/m2 UV radiation. Neither ethanol vehicle nor aloe emodin alone induced skin tumors in the absence of UV radiation. In two separate experiments, 20-30% of the mice treated with a combination of UV radiation and ethanol vehicle and 50-67% of the UV-irradiated animals given aloe emodin in ethanol vehicle developed primary cutaneous melanin-containing tumors. The diagnosis of melanoma was established using Fontana silver stain for melanin; these tumors were negative for vimentin and keratin. Melanin-containing melanosomes were observed by transmission electron microscopy in tumors diagnosed as melanomas. Although the mechanism of carcinogenesis in these mice is currently unknown, our findings have led to the development of the first facile murine model for the induction of primary melanoma. This model has the potential to clarify the role of UV radiation in the etiology of malignant melanoma.

Plants, polysaccharides, and the treatment and prevention of neoplasia.

Plants and Fungi have traditionally been the single largest source of lead compounds for the development of therapeutics by the pharmaceutical industry. Currently mushroom and plant polysaccharides brought to attention by Complementary and Alternative medicine, are undergoing scientific analysis and development to prevent and treat cancer, Two classes of saccharides are under investigation-beta glucan polysaccharides as biological response modifiers for the adjuvant treatment of cancer and "Oligosaccharin"-related oligosaccharides for the prevention of sun-induced skin cancer. Beta glucans already in human trials in the Far East will require mechanistic pharmacologic studies and definition of stucture function relationships before they are ready for clinical trials in the West. Other beta glucans that prime natural killer cells for antibody dependent cell-mediated cytotoxicity are approaching clinical trials. Oligosaccharides that downregulate production of immunosuppressive cytokines by ultraviolet radiation injured keratinocytes are promising agents for the prevention of environmental skin cancer.

Inhibition of UV-induced immune suppression and interleukin-10 production by plant oligosaccharides and polysaccharides.

Application of Aloe barbadensis poly/oligosaccharides to UV-irradiated skin prevents photosuppression of delayed-type hypersensitivity (DTH) responses in mice. We tested the hypothesis that these carbohydrates belong to a family of biologically active, plant-derived polysaccharides that can regulate responses to injury in animal tissues. C3H mice were exposed to 5 kJ/m2 UVB from unfiltered FS40 sunlamps and treated with between 1 pg and 10 micrograms tamarind xyloglucans or control polysaccharides methylcellulose or dextran in saline. The mice were sensitized 3 days later with Candida albicans. Tamarind xyloglucans and purified Aloe poly/oligosaccharides prevented suppression of DTH responses in vivo and reduced the amount of interleukin (IL)-10 observed in UV-irradiated murine epidermis. Tamarind xyloglucans were immunoprotective at low picogram doses. In contrast, the control polysaccharides methylcellulose and dextran had no effect on immune suppression or cutaneous IL-10 at any dose. Tamarind xyloglucans and Aloe poly/oligosaccharides also prevented suppression of immune responses to alloantigen in mice exposed to 30 kJ/m2 UVB radiation. To assess the effect of the carbohydrates on keratinocytes, murine Pam212 cells were exposed to 300 J/m2 UVB radiation and treated for 1 h with tamarind xyloglucans or Aloe poly/oligosaccharides. Treatment of keratinocytes with immunoprotective carbohydrates reduced IL-10 production by approximately 50% compared with the cells treated with UV radiation alone and completely blocked suppressive activity of the culture supernatants in vivo. The tamarind xyloglucans also blocked UV-activated phosphorylation of SAPK/JNK protein but had no effect on p38 phosphorylation. These results indicate that animals, like plants, may use carbohydrates to regulate responses to environmental stimuli.

Aloe barbadensis extracts reduce the production of interleukin-10 after exposure to ultraviolet radiation.

Cutaneous exposure to ultraviolet radiation suppresses the induction of T cell mediated responses such as contact and delayed type hypersensitivity (DTH) by altering the function of immune cells in the skin and causing the release of immunoregulatory cytokines. Extracts of crude Aloe barbadensis gel prevent this photosuppression. Because the regulation of contact hypersensitivity and DTH responses differ, we investigated whether protection was afforded by a single or multiple agents in Aloe and the mechanism by which this material prevents suppression of DTH immunity. The ability of Aloe gel to prevent suppression of contact hypersensitivity responses to hapten decayed rapidly after manufacture. In contrast, agents that protected against systemic suppression of DTH responses to Candida albicans were stable over time. Oligosaccharides prepared from purified Aloe polysaccharide prevented suppression of DTH responses in vivo and reduced the amount of IL-10 observed in ultraviolet irradiated murine epidermis. To assess the effect of Aloe extracts on keratinocytes, Pam 212 cells were exposed in vitro to ultraviolet radiation and treated for 1 h with Aloe oligosaccharides. Culture supernatants were collected 24 h later and injected into mice. Supernatants from ultraviolet irradiated keratinocytes suppressed the induction of DTH responses, whereas Aloe oligosaccharide treatment reduced IL-10 and blocked the suppressive activity of the supernatants. These results indicate that Aloe contains multiple immunoprotective factors and that Aloe oligosaccharides may prevent ultraviolet induced suppression of DTH by reducing keratinocyte derived immunosuppressive cytokines.

Prevention of ultraviolet radiation-induced suppression of contact and delayed hypersensitivity by Aloe barbadensis gel extract.

We investigated the ability of Aloe barbadensis gel extract to prevent suppression of contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH) responses in mice by ultraviolet (UV) irradiation. Local immune suppression was induced in C3H mice by exposure to four daily doses of 400 J/m2 UV-B (280-320 nm) radiation from FS40 sunlamps, followed by sensitization with 0.5% fluorescein isothiocyanate (FITC) through the irradiated skin. Topical application of 0.167-1.67% Aloe gel after each irradiation significantly reduced this suppression. Aloe treatment partially preserved the number and morphology of Langerhans and Thy-1+ dendritic epidermal cells in skin, compared to those in the skin of mice given only UVR or UVR plus the vehicle. Experiments using a single (2 kJ/m2) dose of UVR followed by Aloe treatment showed that the effect of Aloe was not due to screening of the UVR. Systemic suppression of DTH to Candida albicans or CHS to FITC was induced in C3H mice exposed to 5 or 10 kJ/m2 UV-B radiation, respectively, on shaved dorsal skin and sensitized 3 d later with a subcutaneous injection of formalin-fixed Candida or FITC painted on unirradiated, ventral skin. Treatment of the UV-irradiated skin with Aloe immediately after irradiation prevented suppression of both DTH to Candida and CHS to FITC. Aloe treatment did not prevent the formation of cyclobutyl pyrimidine dimers in the DNA of UV-irradiated skin or accelerate the repair of these lesions. These studies demonstrate that topical application of Aloe barbadensis gel extract to the skin of UV-irradiated mice ameliorates UV-induced immune suppression by a mechanism that does not involve DNA damage or repair.

Antibodies to silicone elastomers and reactions to ventriculoperitoneal shunts.

Silicone elastomers used to make medical implants and prostheses are generally believed to be biologically inert. However, we have seen two patients who showed severe, apparently immunemediated, reactions to ventriculoperitoneal (VP) shunts. We used an enzyme-linked immunosorbent assay in which Silastic tubing served as the solid-phase antigen to test serum from the two patients, five other VP shunt patients without inflammatory reactions, and nine healthy adults. IgG binding to Silastic tubing was consistently higher in the two patients than in the healthy or patient controls. The IgG seemed to be binding specifically, since IgG Fab fragments also bound to the tubing, and preincubation of serum with Silastic or silylated proteins removed most of the activity. These findings show that specific immune reactivity to elastomers of polydimethylsiloxane can develop in human beings.

Synergistic action of two oxysterols in the lowering of HMG-CoA reductase activity in CHO-K1 cells.

3 beta-Hydroxy-5 alpha-cholest-8(14)-en-15-one (I) and (25R)-26-hydroxycholesterol (II), both potent regulators of sterol biosynthesis, have been found to show synergism in the reduction of the levels of HMG-CoA reductase activity in CHO-K1 cells. When equimolar concentrations of I and II were added in combination, synergistic reduction (p less than 0.0001) of enzyme activity was observed at total oxysterol concentrations of 0.1 microM, 0.2 microM, and 0.5 microM. Maximal synergistic effect in the lowering of reductase activity (28% greater than predicted) was observed at 0.1 microM total oxysterol concentration. Five additional experiments conducted with 50 nM oxysterols confirmed the synergistic effect at 0.1 microM total sterol concentration. These results suggest that the in vivo importance of I and II may be greater than that anticipated on the basis of the concentrations of the individual sterols.

Inhibitors of sterol synthesis. Morphological studies in rats after dietary administration of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a potent hypocholesterolemic compound.

The morphological effects of short-term (9 days) dietary administration (0.1% in a laboratory chow diet) of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one, a novel regulator of cholesterol metabolism with significant hypocholesterolemic activity, has been studied in young male rats. Control animals included rats fed the basal diet ad libitum and a series of rats pair-fed to the individual experimental animals. At the time of necropsy, the morphological changes in rats which have been observed in rats following treatment with other absorbable hypolipidemic agents (myeloid bodies with triparanol, increased peroxisomes with clofibrate, and proliferation of smooth endoplasmic reticulum with compactin and mevinolin) were not apparent on ultrastructural examination of livers of rats treated with the 15-ketosterol. Two changes were observed in the rats fed the 15-ketosterol: a decrease in adipose tissue and enlargement of the small intestine. Diminished fat was also noted in the pair-fed controls and was presumably due to decreased food consumption. The intestines of rats fed the 15-ketosterol were morphometrically most enlarged in the jejunal region. Morphologically, this increase was distinguished by increased depth of crypts of Lieberkuhn and pseudostratification of epithelium at the base of the villi. These changes were qualitatively and quantitatively similar to the adaptive changes reported in the rat after resection of small bowel or following intestinal bypass (segment of bowel remaining in continuity). The morphological changes induced in the rat by administration of the 15-ketosterol were not observed in 4 baboons which received the compound orally at doses of 50, 75, or 100 mg per kilogram of body weight for up to 3 months.

Comparison of an enzyme-linked immunosorbent assay using crude S. haematobium soluble egg antigen with a radioimmunoassay using purified S. mansoni egg antigen for the serodiagnosis of schistosomiasis haematobium.

An enzyme-linked immunosorbent assay (ELISA) using crude Schistosoma haematobium soluble egg antigen (ShSEA) was compared with a radioimmunoassay (RIA) employing purified heterologous species S. mansoni egg antigen (MSA1) for the serodiagnosis of schistosomiasis haematobium in a group of 45 Nigerian school children living in an area endemic for S. haematobium. Both assay systems appeared applicable. The ELISA proved to be more sensitive detecting 100% of the 27 parasitologically positive individuals while the RIA defined 89% of this group. Neither test had false-positive results for the 10 non-endemic area parasitologically negative controls, although the ELISA demonstrated that 56% of the 18 endemic area parasitologically negative controls had anti-ShSEA antibodies. The RIA indicated that 44% of this group had anti-MSA1 antibodies. These latter findings were interpreted as related to the hyperendemicity of schistosomiasis haematobium for the study area.

Interaction between bovine casein and V. cholerae enterotoxin in the rabbit ileal loop.

Secretory IgA from human breast milk neutralizes cholera enterotoxin in the rabbit ileal loop system. No similar protection by purified bovine milk proteins could be demonstrated; however, one bovine milk protein, casein, had a deleterious effect on intestine exposed to very small quantities of enterotoxin. Highly purified cholera toxin (10 or 100 ng) was incubated with bovine protein solutions for 60 min at 37 degrees C. One-milliliter aliquots were then injected into prepared rabbit intestine loops. The animals were sacrificed at 18 h and the intestinal loop contents were aspirated, and a volume to length of loop ratio (V/L) was determined. The activity of 100 ng of toxin was not enhanced by the majority of bovine milk proteins, but bovine casein caused a 14-40% increase in the fluid production (V/L of casein + toxin versus toxin, 1.05 versus 0.92 and 1.82 versus 1.30). All of the bovine proteins but casein inhibited the action of low dose enterotoxin. Bovine casein caused a 78-90% increase in fluid production by loops exposed to a suboptimal toxin dose (10 ng) (V/L of casein + toxin versus toxin, 1.12 versus 0.63 and 0.95 versus 0.50). Virtually all of this enhancement of enterotoxin fluid response resided in the purified alpha-casein fraction.

Biocompatibility of silicone implants.

Polydimethylsiloxane has been considered immunologically inert, and previous work seems to have established that the production of circulating antibodies does not occur in response to its implantation. We have investigated the possibility of a cellular immune response to implanted silicone. We have observed histologically that the cellular response to polydimethylsiloxane in sensitized guinea pigs is consistent with a cellular immune reaction. Further studies with EM and XES have demonstrated intracellular silicon in the Golgi apparatus, rough endoplasmic reticulum, and at both ends of cytoplasmic bridges between macrophages and lymphocytes. All of these findings fit with the hypothesis that the cells are processing a silicon-containing complex as an antigen. Finally, macrophage migration inhibition studies have shown evidence of a cellular immune phenomenon. Further studies are planned to characterize the nature of the sensitizing complex and to attempt to confirm the migration inhibition studies in vivo.

Efficacy of purified schistosoma japonicum egg antigens for ELISA serodiagnosis of human Schistosomiasis japonica: specificity and sensitivity.

At present, there is no consensus that purified schistosome egg antigens offer any advantage in the diagnosis of schistosomiasis by enzyme linked immunosorbent assay (ELISA). Previously, we demonstrated by multiple techniques that the major serologic antigens in Schistosoma japonicum soluble egg antigen (SEA) are glycoproteins, and that the glycoproteins with highest specificity and sensitivity are hydrophobes. We therefore tested these materials for their specificity, sensitivity and cost effectiveness in the ELISA. In this study we used five SEA fractions that varied in their purity and antigenicity. The order of immunologic specific activity in the ELISA, measured by titration of a standard sera pool, was: hydrophobic glycoproteins (highest), crude SEA glycoproteins, hydrophilic glycoproteins, crude SEA, and SEA proteins (lowest). Complexity (purity) of these materials were (in rank order), hydrophilic glycoproteins (purest), hydrophobic glycoproteins, crude glycoproteins, SEA proteins, and crude SEA (most complex). Epidemiologic sensitivity in the ELISA was tested on limited but well characterized populations. At high antigen coating concentration (0.5 microgram/well), the only antigen fraction with poor sensitivity was SEA proteins. There was little difference in epidemiologic sensitivity between the purer fractions with highest immunologic sensitivity (hydrophobic glycoproteins and crude SEA glycoproteins) and the crude SEA which possesses intermediate immunologic sensitivity. Differences in epidemiologic sensitivity were most pronounced when wells were coated at an antigen concentration (0.1 microgram/well) where crude SEA began to fail. Specificity for all preparations, assessed by reactivity with sera from patients with other trematode infections and with cestode and nematode infections, was excellent. The clinical sensitivity of the ELISA employing crude S. japonicum SEA is so high, and the specificity so good, that the increased immunologic sensitivity of partially purified antigens had little effect on epidemiologic sensitivity. This is not true for the S. mansoni ELISA where crude antigens had inferior sensitivity and specificity.

Fractionation of Schistosoma japonicum soluble egg antigen glycoproteins by hydrophobic interaction chromatography.

We are currently studying the soluble egg antigens of Schistosoma japonicum in an attempt to determine which antigens are potent immunogens. Previously, we demonstrated by Ouchterlony immunodiffusion and inhibition of the circumoval precipitin test that the glycoprotein fraction of soluble egg antigens contains the antigens which are most immunogenic in natural infections. The soluble egg antigen glycoproteins have now been further fractionated via hydrophobic interaction chromatography on phenyl Sepharose. We found that there were at least two antigens involved in the circumoval precipitin reaction. Both the hydrophilic antigen which we call japonicum antigen glycoprotein II (JAG II) and a mixture of hydrophobic antigens (JAG III and the JAG IV complex) were capable of causing a 50% inhibition of the COP reaction around S. japonicum eggs. JAG II was not a major serological antigen of S. japonicum since it gave only a weak precipitin line upon Ouchterlony immunodiffusion analysis with pooled sera from Filipino patients with chronic S. japonicum infections. Hydrophobic interaction chromatography yielded preparations which were sufficiently pure for use in radioimmunoassays. By radioimmunoassay, the best antigens among the glycoproteins were moderately hydrophobic JAG III and the JAG IV complex. They had large amounts of antibody directed toward them in patients with schistosomiasis japonica and exhibited little reactivity with S. mansoni. The hydrophilic glycoproteins JAG I and II were poor immunogens and extensively cross-reacted with S. mansoni. This cross-reactivity means that diagnostic tests with crude soluble egg antigens would run the risk of potential false-negative results in patients with other trematode infections.

Characterization of the glycoprotein antigens which mediate the Schistosoma japonicum circumoval precipitin reaction.

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of (125)I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.

Polymyositis, prednisone, and pheomycotic cyst.

An immunosuppressed patient with polymyositis presented with an apparent ganglion of the left foot. During the operative procedure, a cystic mass inconsistent with a ganglion was excised and immediately sent to the Quantitative Bacteriology Laboratory. A rapid slide examination revealed yeastlike bodies present in the tissue. The remainder of the tissue was sent to pathology for special staining. The H&E and GMS stains revealed findings compatible with the diagnosis of a pheomycotic cyst, and the appropriate cultures confirmed this. This represents an unusual opportunistic infection in an immunosuppressed host. As more patients are managed with immunosuppressive drugs, this diagnosis will need to be considered much more frequently if treatment is to be effective.

Inheritance of mixed cryoglobulinemia.

This paper describes a family in which 10 members of 3 generations have IgM-IgG cryoglobulinemia. Their pedigree is characteristic of autosomal dominant inheritance. No underlying disease that could account for the cryoglobulinemia has been identified in any patient, and no linkage of the cryoglobulinemia to HLA-A and -B locus haplotypes, blood group antigens, or immunoglobulin Gm allotypes has been detected. The rheumatoid factors of this kindred react with some, but not all, human IgG; however, their rheumatoid factors are not antibodies to any known human Gm or Km allotype. This family demonstrates that "essential" mixed cryoglobulinemia can be inherited, and that the clinical manifestations of an inherited cryoglobulinemia may vary among family members.

A shared cryoglobulin antigen in familial cryoglobulinemia.

Ten members of 3 generations of a family have IgM-IgG cryoglobulins and rheumatoid factors in their sera; one additional member has rheumatoid factor but not cryoglobulins. The disorder occurs in an autosomal dominant pattern. Here we describe an antigen, first identified on the cryoglobulin IgM of the index case, which is present in the sera of all 11 members of this kindred with rheumatoid factor. This antigen has the serologic properties of an IgM rheumatoid factor idiotype.