Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Induction of apoptosis in a macrophage cell line RAW 264.7 by acemannan, a beta-(1,4)-acetylated mannan.

Acemannan is a polydispersed beta-(1,4)-linked acetylated mannan with antiviral properties. It is an immunomodulator, and studies in our laboratory have shown that it causes activation of macrophages. In the presence of IFNgamma, acemannan induced apoptosis in RAW 264. 7 cells. These cells exhibited chromatin condensation, DNA fragmentation, and laddering characteristic of apoptosis. The induction of apoptosis by acemannan and IFNgamma does not seem to be mediated by nitric oxide, since N-nitro-L-arginine methyl ester, the nitric oxide inhibitor, had no effect. Acemannan in the presence of IFNgamma also inhibited the expression of bcl-2. These results suggest that acemannan in the presence of IFNgamma induces apoptosis in RAW 264.7 cells through a mechanism involving the inhibition of bcl-2 expression.

Activation of a mouse macrophage cell line by acemannan: the major carbohydrate fraction from Aloe vera gel.

Acemannan is the name given to the major carbohydrate fraction obtained from the gel of the Aloe vera leaf. It has been claimed to have several important therapeutic properties including acceleration of wound healing, immune stimulation, anti-cancer and anti-viral effects. However, the biological mechanisms of these activities are unclear. Because of this wide diversity of effects, it is believed that they may be exerted through pluripotent effector cells such as macrophages. The effects of acemannan on the mouse macrophage cell line, RAW 264.7 cells were therefore investigated. It was found that acemannan could stimulate macrophage cytokine production, nitric oxide release, surface molecule expression, and cell morphologic changes. The production of the cytokines IL-6 and TNF-alpha were dependent on the dose of acemannan provided. Nitric oxide production, cell morphologic changes and surface antigen expression were increased in response to stimulation by a mixture of acemannan and IFN-gamma. These results suggest that acemannan may function, at least in part, through macrophage activation.

Acemannan, a beta-(1,4)-acetylated mannan, induces nitric oxide production in macrophage cell line RAW 264.7.

Acemannan is a polydispersed beta-(1,4)-linked acetylated mannan with antiviral properties. It is an immunomodulator, and studies in our laboratory have shown that it causes activation of macrophages. Inducible NO synthase is generally expressed after transcriptional induction and is known to mediate some of the cytotoxic action of activated macrophages. Acemannan, in the presence of interferon-gamma, greatly increased the synthesis of NO in RAW 264.7 cells. This increase was preceded by increased expression of mRNA for the inducible form of macrophage NO synthase. Preincubation with pyrrolidine dithiocarbamate inhibited the induction, indicating the involvement of nuclear factor-kappa B. These results suggest that acemannan causes the activation of macrophages by increasing the level of NO synthase at the level of transcription.

The effect of Acemannan Immunostimulant in combination with surgery and radiation therapy on spontaneous canine and feline fibrosarcomas.

Eight dogs and five cats with histopathologically confirmed fibrosarcomas were treated with Acemannan Immunostimulanta in combination with surgery and radiation therapy. These animals had recurring disease that had failed previous treatment, a poor prognosis for survival, or both. Following four to seven weekly acemannan treatments, tumor shrinkage occurred in four (greater than 50%; n = 2) of 12 animals, with tumors accessible to measurement. A notable increase in necrosis and inflammation was observed. Complete surgical excision was performed on all animals between the fourth and seventh week following initiation of acemannan therapy. Radiation therapy was instituted immediately after surgery. Acemannan treatments were continued monthly for one year. Seven of the 13 animals remain alive and tumor-free (range, 440+ to 603+ days) with a median survival time of 372 days. The data suggests that Acemannan Immunostimulant may be an effective adjunct to surgery and radiation therapy in the treatment of canine and feline fibrosarcomas.

Pilot study of the effect of acemannan in cats infected with feline immunodeficiency virus.

Acemannan, a complex carbohydrate shown to stimulate interleukin-1, tumor necrosis factor alpha and prostaglandin E2 production by macrophages, has also demonstrated antiviral activity in vitro against human immunodeficiency virus, Newcastle disease virus and influenza virus. A pilot study was undertaken to determine acemannan's effect in 49 feline immunodeficiency virus (FIV) infected cats with clinical signs of disease (Stage 3, 4 or 5), 23 of which had severe lymphopenia. Cats received acemannan either by intravenous (Group 1) or subcutaneous (Group 2) injection once weekly for 12 weeks, or by daily oral (Group 3) administration for 12 weeks. Upon entry into the study, cats were randomly assigned to one of the three groups. Laboratory analyses were performed at the beginning of the study and at Weeks 6 and 12. Cats were allowed to continue with a predetermined maintenance regimen of acemannan after completing the 12-week study. Thirteen cats died during the course of treatment. Upon necropsy, the most frequent histopathologic findings were neoplastic, kidney and pancreatic disease. Friedman's two-way ANOVA test showed no significant differences in efficacy among groups administered acemannan by the different routes. Therefore, groups were combined and a signed-ranks test was used to determine changes over time. A significant increase was seen in lymphocyte counts (P < 0.001). Neutrophil counts decreased significantly (P = 0.007), as did incidence of sepsis (P = 0.008). When cats entering with lymphopenia were analyzed separately, a much greater increase in lymphocyte counts was noted (235%) compared with non-lymphopenic cats (42%). A survival rate of 75% was found for all three groups. Thirty-six of 49 animals are alive 5-19 months post-entry. These results suggest that acemannan therapy may be of significant benefit in FIV-infected cats exhibiting clinical signs of disease.

Antigen dependent adjuvant activity of a polydispersed beta-(1,4)-linked acetylated mannan (acemannan).

The adjuvant properties of a polydispersed beta-(1,4)-linked acetylated mannan, acemannan (ACE-M), were evaluated. Day-old broiler chicks were randomly selected and allocated to four flocks (Vac 1-4). The Vac 1 flock was sham vaccinated with saline. The Vac 2 flock was vaccinated with an oil-based vaccine (Breedervac III; Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious bronchitis virus). The Vac 3 flock was vaccinated with a vaccine-ACE-M mixture, and the Vac 4 flock was vaccinated with vaccine and ACE-M at separate anatomical sites. ELISA titres to NDV and IBDV were determined. The immune response to NDV at 21 days postvaccination (PV) was significantly enhanced (P less than or equal to 0.05) by the addition of ACE-M to the vaccine, compared with vaccination without ACE-M. Subsequently, the vaccine-ACE-M mixture appeared to suppress the immune response to NDV. However, at day 35 PV, 95% of the Vac 3 chicks compared with 90% of the Vac 2 and 89% of the Vac 4 chicks exhibited protective titres. The response to IBDV differed from that to NDV. At day 21 PV the immune response to IBDV was essentially the same for all flocks that received vaccine, i.e. addition of ACE-M to the vaccine did not significantly enhance the immune response; however, it did significantly (P less than or equal to 0.05) sustain the immune response at days 28 and 35. In addition to the observed effect on titres to NDV and IBDV, ACE-M also had an effect on flock immunity.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of the effect of acemannan on retrovirus infections: clinical stabilization of feline leukemia virus-infected cats.

Feline leukemia is a disease induced by an oncornavirus infection that inevitably causes clinically affected cats to die. It has been estimated that 40% of cats are dead within 4 weeks and 70% within 8 weeks of the onset of clinical symptoms. Acemannan is a complex carbohydrate with both immunostimulatory and direct antiviral properties. Administration of acemannan for 6 weeks intraperitoneally to clinically symptomatic cats significantly improved both the quality of life and the survival rate. Twelve weeks after initiation of treatment, 71% of treated cats were alive and in good health.

The biological activities of mannans and related complex carbohydrates.

Complex polymers containing mannose (mannans) possess significant biological activity when administered to mammals. When given orally, they inhibit cholesterol absorption and induce hypocholesterolemia. If administered by other routes, they bind to mannose-binding proteins and induce macrophage activation and interleukin-1 release, inhibit viral replication, stimulate bone marrow activity, promote wound healing and inhibit tumor growth. This range of activities makes the mannans, potentially important biological-response modifiers and therapeutic agents.

Serological cross-reactions between Brucella abortus and Yersinia enterocolitica 0:9.

Yersinia enterocolitica (Ye) 0:9 is an organism of great significance in veterinary medicine largely as a result of its cross-reaction with Brucella abortus (Ba). Boty Ye 0:9 and Ba possess somatic antigens in common; as a result of which animals exposed to Ye 0:9 have an immune response which is distinguishable only with difficulty from that induced by Ba. Cattle were exposed to Ye 0:9 by the oral or intramammary routes. Oral exposure failed to generate significant serologic response. In contrast, intramammary inoculation produced a marked response. Serum antibodies provoked in this manner reacted strongly with Ba. The anti-Brucella response provoked by inoculation of Yersinia was sufficient to render milk and serum Brucella-seropositive as measured by the standard milk ring and serum agglutination tests. While both Ba and Ye 0:9 have 9 antigens in common, they differ significantly with respect to motility. Thus Ba is always non motile while Ye is motile when grown at room temperature. The presence of Yersinia H agglutinins in serum were shown to be evidence of previous exposure to Ye. The H agglutinins were not generated by Brucella infection. A rapid H agglutination test was shown to provide this differentiation without interference from cross-reacting O antigens. Results of Ba O and Ye O and OH antigens used in the agglutination test were found useful to differentiate antibodies against Ba from those induced by Ye 0:9 in cattle sera. The existence of enterobacterial common antigen (ECA) in Ye and its absence in Ba were utilized in an attempt to provide a method to distinguish Brucella infections from those with cross-reacting Yersinia.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of phospholipase A1 from Trypanosoma brucei.

Phospholipase A1 from Trypanosoma brucei brucei has been purified 380-fold by column chromatography on phosphatidylcholine-Sepharose affinity columns followed by DEAE-cellulose anion-exchange chromatography and Sephacryl S-200 molecular exclusion chromatography. Octyl-Agarose hydrophobic column chromatography can be substituted for the PC-Sepharose column. The molecular weight of trypanosomal PLase A1 was found to be 26,000 by SDS-polyacrylamide gel electrophoresis.

Agglutination tests and their modifications in the diagnosis of bovine brucellosis.

This article reviews the effects of using Yersinia enterocolitica serotype: 09 somatic antigen in diagnostic tests for brucellosis. The tests employed include the standard tube agglutination test, the microplate agglutination test, the quantitative plate agglutination test, the growth agglutination test and the indirect hemagglutination technique. Only the growth agglutination technique demonstrated an increased sensitivity over the STAT. Neither the microplate agglutination test nor quantitative plate tests offered any significant advantages over the STAT. The QPAT using Brucella O antigen was clearly subject to false negative results. The indirect hemagglutination technique showed extreme variation in results, the significance of which (if any) was unclear.

False-Positive Reactions in the Immunoprecipitation Test for Meat Identification.

During the course of experiments into the specificity of the ring precipitation test it was found that a commercial anti-porcine serum reacted with bovine plasma. When this phenomenon was investigated it was found to be non-immunological in nature and that plasma obtained from blood treated with calcium chelating agents gave a positive reaction with normal serum. The species of origin of the reactants appeared to have no effect on the reaction. Further analysis indicated that a similar false-positive reaction could be obtained using sodium ascorbate either as an anticoagulant or as an additive to ground beef. Preliminary studies were conducted on the mechanism of this reaction.

Lysophospholipase 1 in Trypanosoma brucei.

Protein fractions from Trypanosoma brucei brucei showed lysophospholipase 1 activity (E.E.3.1.1.5), against the substrate 1-acyl-sn-glycero-3-phosphocholine, and also phospholipase A1 activity (E.C.3.1.1.4) by hydrolysis of the 1-acyl bond of 1,2-diacyl-sn-glycero-3-phosphocholine. Both enzyme activities were eluted together and showed a 12-fold purification following Sephacryl S-200 column chromatography. A final 96-fold increase in activity was obtained by electrophoresis on nondenaturing polyacrylamide gels to yield a band containing both enzymic activities. Phospholipase A1 showed maximum activity between pH 6.0--8.5 and lysophospholipase 1 had a pH optimum of 8.5. Both activities were found mainly in the soluble fraction of disrupted trypanosomes and were similarly inhibited by N-ethylmaleimide and p-chloromercuribenzoic acid. Although Triton X-100 stimulated phospholipase A1 activity, it inhibited lysophospholipase 1 activity. The Km value for the lysophospholipase 1 was found to be 0.15 mM. It was not possible to resolve separate activities for lysophospholipase 1 and phospholipase A1 and the ratio of the two activities was approximately 1 : 10 for a variety of preparations and treatments. It is probable that a single enzyme displays both activities.

Serologic response of pigs to experimental infection with Yersinia enterocolitica serotype O9 and Brucella abortus.

Groups of young pigs were inoculated with either Brucella abortus or Yersinia enterocolitica O:9 and their serum was analyzed to identify techniques by which infections with these organisms could be distinguished. Yersinia enterocolitica was capable consistently of inducing a significant anti-Brucella agglutinin response. By means of microplate agglutination, it was shown that homologous titers usually exceeded or were equal to heterologous titers. This technique was also capable of detecting Yersinia flagellar agglutinins. When a group of serum samples from healthy pigs was surveyed, it was found that none of the techniques tested were capable of unequivocally distinguishing between the 2 infections.

Experimental intramammary infection of cows with Yersinia enterocolitica O9: cellular and immunologic responses.

Three clinically normal cows were inoculated with viable Yersinia enterocolitica O9 by the intramammary route. This provoked an increase in the somatic cell count in milk from the inoculated quarter, but no obvious clinical response. Yersinia enterocolitica was isolated only sporadically from these cattle after inoculation. All cows showed a marked serologic response to Y enterocolitica and Brucella abortus. The anti-Brucella response provoked by inoculation of Yersinia was sufficient to render milk and serum Brucella-seropositive, as measured by the standard milk ring test, the quantitative milk ring test, and the serum agglutination test.

The phospholipases of pathogenic and non-pathogenic Trypanosoma species.

Four species of trypanosome were examined for phospholipase activities using 1-[3H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine and 1-acyl-2[14C]linoleoyl-sn-glycero-3-phosphocholine as substrates. The major activity in each species is a phospholipase A1 (EC 3.1.1.32) which does not require calcium. The most effective of the detergents tested for activation of the enzyme from each species, and the Ph optima, are as follows: Trypanosoma brucei, 0.125% Triton X-100 at pH 6.0-8.5; T. congolense, 0.5 mM linoleate at pH 6.0; T. theileri, 0.1% Triton X-100 at pH 6.75; T. lewisi, 0.2 mM sodium dodecyl sulfate at pH 5.2. The specific activity of the enzyme from a pathogenic species, T. brucei, is very high (145 nmol/min/mg/protein) and could contribute to the tissue damage characteristically caused by this parasite. The level in T. lewisi, a non-pathogenic species, is relatively low (1 nmol/min/mg). The levels in T. theileri (31 nmol/min/mg) and T. congolense (10 nmol/min/mg are intermediate. These results are compatible with the hypothesis that phospholipases contribute to the pathogenicity of trypanosomes.

A standardized enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Toxoplasma gondii.

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed. This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents. In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficient of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.

Accumulation of phospholipase A1 in tissue fluid of rabbits infected with Trypanosoma brucei.

Samples of tissue fluid were obtained from plastic cages implanted subcutaneously in rabbits. A phospholipase A1 similar to that found in Trypanosoma brucei appeared in the tissue fluid about seven days after infection with this parasite, increasing with parasite burden, and reaching levels of more than 5 nmol phosphatidylcholine hydrolysed per min per ml tissue fluid. The large amount of phospholipase A1 found free in the tissue fluid appeared to be of trypanosomal origin and was either secreted by living parasites or released from dying organisms. Phospholipase A1 was detectable in blood plasma from the infected rabbits, but at a level considerably lower than in the tissue fluid. An inhibitor of the phospholipase was present in the plasma after the first two week of infection which may be partly responsible for this lower level.