Production of recombinant antigens in plants for animal and human immunization - a review

Plants present a cost effective production system for high value proteins. There is an increasing world demand for cheap vaccines that can be readily administered to the population, especially in economically less developed regions. A promising concept is the production of vaccines in plants that could be grown locally. Expression of antigenic peptides in the palatable parts of plants can lead to the production of edible active vaccines. Two major strategies are: i) to express antigens in transgenic plants, and ii) to produce antigenic peptides on the surface of plant viruses that could be used to infect host plants. This review considers the experimental data and early results for both strategies, and discusses the potential and problems of this new technology

Animal models of anxiety: an ethological perspective

In the field of anxiety research, animal models are used as screening tools in the search for compounds with therapeutic potential and as simulations for research on mechanisms underlying emotional behaviour. However, a solely pharmacological approach to the validation of such tests has resulted in distinct problems with their applicability to systems other than those involving the benzodiazepine/GABAA receptor complex. In this context, recent developments in our understanding of mammalian defensive behaviour have not only prompted the development of new models but also attempts to refine existing ones. The present review focuses on the application of ethological techniques to one of the most widely used animal models of anxiety, the elevated plus-maze paradigm. This fresh approach to an established test has revealed a hitherto unrecognized multidimensionality to plus-maze behaviour and, as it yields comprehensive behavioural profiles, has many advantages over conventional methodology. This assertion is supported by reference to recent work on the effects of diverse manipulations including psychosocial stress, benzodiazepines, GABA receptor ligands, neurosteroids, 5-HT1A receptor ligands, and panicolytic/panicogenic agents. On the basis of this review, it is suggested that other models of anxiety may well benefit from greater attention to behavioural detail

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropism and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of Old World and New World monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK) by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8) and females 29.3 ± 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8) and females 26.1 ± 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6) and females 1.18 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6) and females 1.34 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Bi-directional actions of estrogen on the renin-angiotensin system

Estrogen stimulates the renin-angiotensin system by augmenting both tissue and circulating levels of angiotensinogen and renin. We show, however, that angiotensin converting enzyme (ACE) activity in the circulation and in tissues is reduced in two animal models of postmenopausal chronic hormone replacement. We observed a reduction of ACE activity in association with a significant increase in plasma angiotensin I (Ang I) and hyperreninemia in ovariectomized monkeys treated with Premarin (conjugated equine estrogen) replacement for 30 months. Plasma angiotensin II (Ang II) levels were not increased in monkeys treated with estrogen, suggesting that the decrease in ACE curtailed the formation of the peptide. The Ang II/Ang I ratio, an in vivo index of ACE activity, was significantly reduced by estrogen treatment, further supporting the biochemical significance of estrogen's inhibition of ACE. In ovariectomized transgenic hypertensive (mRen2)27 rats submitted to estrogen replacement treatment for 3 weeks, ACE activity in plasma and tissue (aorta and kidney) and circulating Ang II levels were reduced, whereas circulating levels of angiotensin-(1-7) (Ang-(1-7) were increased. Ang-(1-7), the N-terminal fragment of Ang II, is a novel vasodilator and antihypertensive peptide. Thus, the net balance of these effects of estrogen on the renin-angiotensin vasoconstrictor/vasodilator system is to promote the antihypertensive effect.

Heparan sulfates and heparins: similar compounds performing the same functions in vertebrates and invertebrates?

The distribution and structure of heparan sulfate and heparin are briefly reviewed. Heparan sulfate is a ubiquitous compound of animal cells whose structure has been maintained throughout evolution, showing an enormous variability regarding the relative amounts of its disaccharide units. Heparin, on the other hand, is present only in a few tissues and species of the animal kingdom and in the form of granules inside organelles in the cytoplasm of special cells. Thus, the distribution as well as the main structural features of the molecule, including its main disaccharide unit, have been maintained through evolution. These and other studies led to the proposal that heparan sulfate may be involved in the cell-cell recognition phenomena and control of cell growth, whereas heparin may be involved in defense mechanisms against bacteria and other foreign materials. All indications obtained thus far suggest that these molecules perform the same functions in vertebrates and invertebrates.

The relationship between anxiety and depression in animal models: a study using the forced swimming test and elevated plus-maze

The present study evaluated the correlation between the behavior of mice in the forced swimming test (FST) and in the elevated plus-maze (PM). The effect of the order of the experiments, i.e., the influence of the first test (FST or PM) on mouse behavior in the second test (PM or FST, respectively) was compared to handled animals (HAND). The execution of FST one week before the plus-maze (FST-PM, N = 10), in comparison to mice that were only handled (HAND-PM, N = 10) in week 1, decreased % open entries (HAND-PM: 33.6 ± 2.9; FST-PM: 20.0 ± 3.9; mean ± SEM; P<0.02) and % open time (HAND-PM: 18.9 ± 3.3; FST-PM: 9.0 ± 1.9; P<0.03), suggesting an anxiogenic effect. No significant effect was seen in the number of closed arm entries (FST-PM: 9.5 (7.0-11.0); HAND-PM: 10.0 (4.0-14.5), median (interquartile range); U = 46.5; P>0.10). A prior test in the plus-maze (PM-FST) did not change % immobility time in the FST when compared to the HAND-FST group (HAND-FST: 57.7 ± 3.9; PM-FST: 65.7 ± 3.2; mean ± SEM; P>0.10). Since these data suggest that there is an order effect, the correlation was evaluated separately with each test sequence: FST-PM (N = 20) and PM-FST (N = 18). There was no significant correlation between % immobility time in the FST and plus-maze indexes (% time and entries in open arms) in any test sequence (r: -0.07 to 0.18). These data suggest that mouse behavior in the elevated plus-maze is not related to behavior in the forced swimming test and that a forced swimming test before the plus-maze has an anxiogenic effect even after a one-week interval.

The brain decade in debate: II. Panic or anxiety? From animal models to a neurobiological basis

This article is a transcription of an electronic symposium sponsored by the Brazilian Society of Neuroscience and Behavior (SBNeC). Invited researchers from the European Union, North America and Brazil discussed two issues on anxiety, namely whether panic is a very intense anxiety or something else, and what aspects of clinical anxiety are reproduced by animal models. Concerning the first issue, most participants agreed that generalized anxiety and panic disorder are different on the basis of clinical manifestations, drug response and animal models. Also, underlying brain structures, neurotransmitter modulation and hormonal changes seem to involve important differences. It is also common knowledge that existing animal models generate different types of fear/anxiety. A challenge for future research is to establish a good correlation between animal models and nosological classification.

Incorporation of dietary trans monounsaturated fatty acids into tissues of Walker 256 tumor-bearing rats

The correlation between dietary trans fatty acids and neoplasia was examined in the present study. Walker 256 tumor-bearing and control rats were fed a trans monounsaturated fatty acid (MUFA)-rich diet for 8 weeks and the incorporation of trans fatty acids by tumor tissue was examined. Also, the effect of tumor growth on trans fatty acid composition of plasma and liver, and the content of thiobarbituric acid-reactive substances (TBARS) was determined. Walker 256 tumor cells presented both trans and cis MUFAs given in the diet. The equivalent diet proportions were 0.66 for trans and 1.14 for cis. Taking into consideration the proportion of trans MUFAs in plasma (11.47%), the tumor incorporated these fatty acids in a more efficient manner (18.27%) than the liver (9.34%). Therefore, the dietary trans fatty acids present in the diet are actively incorporated by the tumor. Tumor growth itself caused marked changes in the proportion of polyunsaturated fatty acids in the plasma and liver but provoked only slight modifications in both trans and cis MUFAs. Tumor growth also reduced the unsaturation index in both plasma and liver, from 97.79 to 86.83 and from 77.51 to 69.64, respectively. This effect was partially related to an increase in the occurrence of the lipid oxidation/peroxidation process of TBARS content which was increased in both plasma (from 0.428 to 0.505) and liver (from 9.425 to 127.792) due to tumor growth.

The DNA puff BhB10-1 gene is differentially expressed in various tissues of Bradysia hygida late larvae and constitutively transcribed in transgenic Drosophila

We extended the characterization of the DNA puff BhB10-1 gene of Bradysia hygida by showing that, although its mRNA is detected only at the end of the fourth larval instar, BhB10-1 expression is not restricted to the salivary gland, the tissue in which this gene is amplified. Different amounts of BhB10-1 mRNA were detected in other larval tissues such as gut, Malpighian tubules, fat body, brain and cuticle, suggesting that this gene is expressed differentially in the various tissues analyzed. Analysis of transgenic Drosophila carrying the BhB10-1 transcription unit and flanking sequences revealed that the tested fragment promotes transcription in a constitutive manner. We suggest that either cis-regulatory elements are missing in the transgene or factors that temporally regulate the BhB10-1 gene in B. hygida are not conserved in Drosophila.

Head-to-head comparison of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia in an animal model of coronary artery stenosis

To compare the sensitivity of dipyridamole, dobutamine and pacing stress echocardiography for the detection of myocardial ischemia we produced a physiologically significant stenosis in the left circumflex artery of 14 open-chest dogs (range: 50 to 89% reduction in luminal diameter). In each study, dobutamine (5 to 40 µg kg-1 min-1 in 3-min stages) and pacing (20 bpm increments, each 2 min, up to 260 bpm) were performed randomly, and then followed by dipyridamole (up to 0.84 mg/kg over 10 min). The positivity of stress echocardiography tests was quantitatively determined by a significant (P<0.05) reduction of or failure to increase absolute and percent systolic wall thickening in the stenotic artery supplied wall, as compared to the opposite wall (areas related to the left anterior descending artery). Systolic and diastolic frozen images were analyzed off-line by two blinded observers in the control and stress conditions. The results showed that 1) the sensitivity of dobutamine, dipyridamole and pacing stress tests was 57, 57 and 36%, respectively; 2) in animals with positive tests, the mean percent change of wall thickening in left ventricular ischemic segments was larger in the pacing (-19 ± 11%) and dipyridamole (-18 ± 16%) tests as compared to dobutamine (-9 ± 6%) (P = 0.05), but a similar mean reduction of wall thickening was observed when this variable was normalized to a control left ventricular segment (area related to the left anterior descending artery) (pacing: -16 ± 7%; dipyridamole: -25 ± 16%; dobutamine: -26 ± 10%; not significant), and 3) a significant correlation was observed between magnitude of coronary stenosis and left ventricular segmental dysfunction induced by ischemia in dogs submitted to positive stress tests. We conclude that the dobutamine and dipyridamole stress tests showed identical sensitivities for the detection of myocardial ischemia in this one-vessel disease animal model with a wide range of left circumflex artery stenosis. The pacing stress test was less sensitive, but the difference was not statistically significant. The magnitude of segmental left ventricular dysfunction induced by ischemia was similar in all stress tests evaluated.

Influence of exercise on the activity and the distribution between free and bound forms of glycolytic and associated enzymes in tissues of horse mackerel

The effects of short-term burst (5 min at 1.8 m/s) swimming and long-term cruiser (60 min at 1.2 m/s) swimming on maximal enzyme activities and enzyme distribution between free and bound states were assessed for nine glycolytic and associated enzymes in tissues of horse mackerel, Trachurus mediterraneus ponticus. The effects of exercise were greatest in white muscle. The activities of phosphofructokinase (PFK), pyruvate kinase (PK), fructose-1,6-bisphosphatase (FBPase), and phosphoglucomutase (PGM) all decreased to 47, 37, 37 and 67%, respectively, during 60-min exercise and all enzymes except phosphoglucoisomerase (PGI) and PGM showed a change in the extent of binding to subcellular particulate fractions during exercise. In red muscle, exercise affected the activities of PGI, FBPase, PFK, and lactate dehydrogenase (LDH) and altered percent binding of only PK and LDH. In liver, exercise increased the PK activity 2.3-fold and reduced PGI 1.7-fold only after 5 min of exercise but altered the percent binding of seven enzymes. Fewer effects were seen in brain, with changes in the activities of aldolase and PGM and in percent binding of hexokinase, PFK and PK. Changes in enzyme activities and in binding interactions with subcellular particulate matter appear to support the altered demands of tissue energy metabolism during exercise.

The role of CD8+ T cells during allograft rejection

Organ transplantation can be considered as replacement therapy for patients with end-stage organ failure. The percent of one-year allograft survival has increased due, among other factors, to a better understanding of the rejection process and new immunosuppressive drugs. Immunosuppressive therapy used in transplantation prevents activation and proliferation of alloreactive T lymphocytes, although not fully preventing chronic rejection. Recognition by recipient T cells of alloantigens expressed by donor tissues initiates immune destruction of allogeneic transplants. However, there is controversy concerning the relative contribution of CD4+ and CD8+ T cells to allograft rejection. Some animal models indicate that there is an absolute requirement for CD4+ T cells in allogeneic rejection, whereas in others CD4-depleted mice reject certain types of allografts. Moreover, there is evidence that CD8+ T cells are more resistant to immunotherapy and tolerance induction protocols. An intense focal infiltration of mainly CD8+CTLA4+ T lymphocytes during kidney rejection has been described in patients. This suggests that CD8+ T cells could escape from immunosuppression and participate in the rejection process. Our group is primarily interested in the immune mechanisms involved in allograft rejection. Thus, we believe that a better understanding of the role of CD8+ T cells in allograft rejection could indicate new targets for immunotherapy in transplantation. Therefore, the objective of the present review was to focus on the role of the CD8+ T cell population in the rejection of allogeneic tissue.