Inactivation of viruses infecting ectothermic animals by amphibian and piscine antimicrobial peptides.

The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.

Effects of agricultural pesticides on the immune system of Xenopus laevis and Rana pipiens.

Over the last 30 years, there have been mass declines in diverse geographic locations among amphibian populations. Multiple causes have been suggested to explain this decline. Among these, environmental pollution is gaining attention. Indeed, some chemicals of environmental concern are known to alter the immune system. Given that amphibians are frequently exposed to agricultural pesticides, it is possible that these pollutants alter their immune system and render them more susceptible to different pathogens. In this study, we exposed two frog species, Xenopus laevis and Rana pipiens, for a short period of time to a mixture of pesticides (atrazine, metribuzine, endosulfan, lindane, aldicarb and dieldrin) representative in terms of composition and concentrations to what it is found in the environment of the southwest region of the province of Quebec. The pesticides were known to be present in surface water of many tributaries of the St. Lawrence River (Quebec, Canada). Our results demonstrate that the mixture of pesticides could alter the cellularity and phagocytic activity of X. laevis and the lymphocyte proliferation of R. pipiens. Taken together, these results indicate that agricultural pesticides can alter some aspects of the immune response in frogs and could contribute to their global decline by rendering them more susceptible to certain infections.

Isolation of peptides of the brevinin-1 family with potent candidacidal activity from the skin secretions of the frog Rana boylii.

The emergence of strains of the human pathogen Candida albicans with resistance to commonly used antibiotics has necessitated a search for new types of antifungal agents. Six peptides with antimicrobial activity were isolated from norepinephrine-stimulated skin secretions from the foothill yellow-legged frog Rana boylii. Brevinin-1BYa (FLPILASLAA10KFGPKLF CLV20TKKC) was particularly potent against C. albicans [minimal inhibitory concentration (MIC) = 3 microm] and also active against Escherichia coli (MIC = 17 microm) and Staphylococcus aureus (MIC = 2 microm), but its therapeutic potential for systemic use is limited by its strong hemolytic activity (HC50 = 4 microm). The single amino acid substitution (Phe12 --> Leu) in brevinin-1BYb resulted in a fourfold lower potency against C. albicans and the additional amino acid substitutions (Lys11 --> Thr, Phe17 --> Leu and Val20 --> Ile) in brevinin-1BYc resulted in a ninefold decrease in activity. Two members of the ranatuerin-2 family and one member of the temporin family were also isolated from the secretions but showed relatively low potency against the three microorganisms tested.

Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin.

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

Neuroendocrine-immune system interactions in amphibians: implications for understanding global amphibian declines.

Amphibians are ancient creatures valued by biologists and naturalists around the world. They share with all other vertebrates a complex neuroendocrine system that enables them to flourish in a variety of aquatic and semiaquatic environments. Studies from a number of laboratories have demonstrated that the immune system of amphibian species is nearly as complex as that of mammals. Yet for reasons that are not well understood, amphibian species are facing greater survival challenges than in the recent past. This article will review our current understanding of the neuroendocrine immune system interactions in amphibians and address the question of whether environmental stressors may contribute to immunosuppression and amphibian declines.

FKBP binding characteristics of cardiac microsomes from diverse vertebrates.

FK506 binding protein (FKBP) is a cytosolic receptor for the immunosuppressive drug FK-506. The common isoform, FKBP12, was found to be associated with the calcium release channel (ryanodine receptor 1) of different species of vertebrate skeletal muscle, whereas 12.6, a novel FKBP isoform was found to be associated with canine cardiac ryanodine receptor (ryanodine receptor 2). Until recently, canine cardiac sarcoplasmic reticulum was considered to be the prototype for studying heart RyR2 and its interactions with FKBP. In this study, cardiac microsomes were isolated from diverse vertebrates: human, rabbit, rat, mice, dog, chicken, frog, and fish and were analyzed for their ability to bind or exchange with FKBP isoforms 12 and 12.6. Our studies indicate that RyR2 from seven out of the eight animals contain both FKBP12 and 12.6. Dog is the exception. It can now be concluded that the association of FKBP isoforms with RyR2 is widely conserved in the hearts of different species of vertebrates.

Hematological and antifungal properties of temporin A and a cecropin A-temporin A hybrid.

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.

Pituitary involvement in T cell renewal during development and metamorphosis of Xenopus laevis.

Studies of pituitary-deficient dwarf mice show that in the absence of a normally functioning pituitary, thymus development is impaired. Treatment with growth hormone, prolactin, and thyroid hormones restores thymus development. Smaller thymus size in pituitary-deficient animals could be due to defective development of precursors, impaired precursor immigration, impaired thymocyte expansion, or development of a smaller epithelial/stromal compartment in the thymus of pituitary-deficient animals. Using a well-characterized amphibian model to study stem cell immigration into the thymus, we show here that hypophysectomy (hypx) of young tadpoles interferes with overall growth of the frogs and with the broad lymphocyte expansion that occurs after metamorphosis, but it does not interfere with the immigration of T cell precursors into an implanted thymus. Diploid host cells moving into a triploid thymus implant do so at the same rate and to the same extent in hypx hosts as they do in intact control hosts. Analysis of cell division in the implanted thymus populations shows a significantly greater proportion of cells arrested in the G(0)/G(1) phase and a significantly lower proportion of cells in the S phase and G(2) + M phase of the cell cycle in hypx hosts than in intact hosts. Thus, smaller thymus size in hypx hosts could be due to a slower rate of expansion of precursors that migrate there.

Amphibian declines: an immunological perspective.

Many, but not all, amphibian populations have been declining on all six continents on which they live. Although habitat destruction, direct application of toxicants, and introduction of predators/competitors are obvious causes of amphibian declines, many amphibians are dying of infectious diseases in relatively pristine habitats on several continents. In this paper, we review the patterns of these disease outbreaks and the characteristics of amphibian immune systems. Hypotheses are presented to explain the apparent susceptibility of amphibians to these pathogens. Natural and man-made factors that can alter amphibian immune responses to pathogens are discussed. Additional research is needed on the biology of the specific pathogens, the pattern of immune responses they elicit, and the nature of environmental stressors that may increase susceptibility to infectious disease.

Metamorphosis and the amphibian immune system.

Studies of the ontogeny of immunity in a limited number of representative amphibians have shown that while the immune systems of the larval forms are competent to defend against potential pathogens in the temporary ponds they inhabit, they are not equivalent to the mature immune systems that develop after metamorphosis. Metamorphosis is a critical time of transition when increased concentrations of metamorphic hormones, principally thyroid hormones (TH) and corticosteroid hormones (CH), orchestrate the loss or reorganization of many tissues and organ systems, including the immune system. Immune system reorganization may serve to eliminate unnecessary lymphocytes that could be destructive if they recognized newly emerging adult-specific antigens on the adult tissues. Increased corticosteroids during metamorphosis appear to induce apoptosis of susceptible lymphocytes. This cell death can be inhibited in vitro or in vivo by the corticosteroid receptor antagonist, RU486. A coordinate increase in both TH and CH at metamorphosis may be common to all amphibians that undergo metamorphosis. Current evidence suggests that the central hypothalamic mediator that induces pituitary production of both thyroid-stimulating hormone and adrenocorticotropic hormone in larval amphibians is corticotropin-releasing hormone. Most amphibians probably survive the temporary immunosuppression associated with metamorphosis with no deleterious effects. However, it is hypothesized that if environmental stressors result in the induction of metamorphosis at a less than optimal body size and state of immune maturation, the immune system destruction would be more significant, and the amphibians could be at greater risk of infection and death.

In vitro studies of spontaneous and corticosteroid-induced apoptosis of lymphocyte populations from metamorphosing frogs/RU486 inhibition.

Metamorphosis in the South African clawed frog, Xenopus laevis, results in significant changes in the immune system. It is characterized by a striking involution of the thymus and spleen followed by lymphocyte expansion in the postmetamorphic period. While thyroid hormones are generally thought to be the most important mediators of morphological changes during metamorphosis, corticosteroid hormones (CH) have also been shown to accelerate metamorphic changes. We have been studying the possible role of CH as effectors of changes in the immune system at metamorphosis. Because CH induce apoptosis of developing murine thymocytes, we examined in vitro levels of spontaneous and CH-inducible apoptosis of lymphocyte populations removed from the thymus and spleen of tadpoles before metamorphosis, during the period of naturally elevated corticosteroids at the climax of metamorphosis, and from postmetamorphic adults. We show here that spontaneous apoptosis of splenocytes or thymocytes measurable at the time of sacrifice or after culture for 24 h at 4 degrees C is very low at all stages of development and is not increased at metamorphosis. Apoptosis induced by culture of lymphocytes for 24 h at 26 degrees C in 10 nM corticosterone (well below the peak level of 70 nM found at climax of metamorphosis) ranges from about 30-50% in the splenocyte population and 55-70% in the thymocyte population. Using the corticosteroid hormone receptor antagonist, RU486, we separated the CH-dependent component of apoptosis from apoptosis due to other factors. In the spleen, about 12-23% of lymphocytes are susceptible to corticosteroid-induced apoptosis at all larval stages as well as during climax of metamorphosis as measured by this short term culture assay. Another approximately 15% of cells undergo spontaneous apoptosis which is independent of CH. Although dissociated thymocytes exhibit very high levels of apoptosis (55-75%) during culture at 26 degrees C for 24 h, most of the apoptosis is independent of CH and may result from loss of "survival signals" due to the disruption of the thymic microenvironment. These studies support the hypothesis that naturally elevated levels of endogenous free CH delete a significant proportion of the larval splenocyte population during climax of metamorphosis by induction of apoptosis. This clearing of lymphocytes may prevent destructive autoimmune responses to the new set of adult-specific antigens that emerges at metamorphosis.

Involvement of thyroid hormones in the expression of MHC class I antigens during ontogeny in Xenopus.

The major histocompatibility complex (MHC) is a cluster of genes encoding products central to all major functions of the vertebrate immune system. Evidence for an MHC can be found in all vertebrate groups that have been examined except the jawless fishes. Expression of MHC class I and class II antigens early in ontogeny is critically important for development of T lymphocytes capable of discriminating self from nonself. Because of this essential role in T-cell development, the ontogeny of MHC expression in the South African clawed frog, Xenopus laevis, was studied. Previous studies of MHC class I expression in Xenopus laevis suggested that class I antigens are virtually absent from tadpole tissues until climax of metamorphosis. We therefore examined the possible role of thyroid hormones (TH) in the induction of class I. By flow cytometry, a small amount of class I expression was detectable on splenocytes and erythrocytes in untreated frogs at prometamorphic stages 55-58, and the amount increased significantly at the conclusion of metamorphic climax. Thus, metamorphosis is associated with increased intensity of class I expression. Neither inhibition nor acceleration of metamorphosis altered the timing of onset of class I expression. However, inhibition of metamorphosis prevented the increase in class I expression characteristic of adult cell populations. Because expression was not accelerated in TH-treated frogs or delayed in metamorphosis-inhibited frogs, it is unlikely that TH are the direct developmental cues that induce expression, although they seem to be required for the upregulation of class I expression occurring at metamorphosis. Differences in the pattern of expression in different sub-populations of cells suggest a complex pattern of regulation of expression of class I antigens during ontogeny.

Involvement of glucocorticoids in the reorganization of the amphibian immune system at metamorphosis.

In recent years, integrative animal biologists and behavioral scientists have begun to understand the complex interactions between the immune system and the neuroendocrine system. Amphibian metamorphosis offers a unique opportunity to study dramatic hormone-driven changes in the immune system in a compressed time frame. In the South African clawed frog, Xenopus laevis, the larval pattern of immunity is distinct from that of the adult, and metamorphosis marks the transition from one pattern to the other. Climax of metamorphosis is characterized by significant elevations in thyroid hormones, glucocorticoid hormones, and the pituitary hormones, prolactin and growth hormone. Previously, we and others have shown that elevated levels of unbound glucocorticoid hormones found at climax of metamorphosis are associated with a natural decline in lymphocyte numbers, lymphocyte viability, and mitogen-induced proliferation. Here we present evidence that the mechanism for loss of lymphocytes at metamorphosis is glucocorticoid-induced apoptosis. Inhibition of lymphocyte function and loss of lymphocytes in the thymus and spleen are reversible by in vitro or in vivo treatment with the glucocorticoid receptor antagonist, RU486, whereas the mineralocorticoid receptor antagonist, RU26752, is poorly effective. These observations support the hypothesis that loss of larval lymphocytes and changes in lymphocyte function are due to elevated concentrations of glucocorticoids that remove unnecessary lymphocytes to allow for development of immunological tolerance to the new adult-specific antigens that appear as a result of metamorphosis.

Late thymectomy in Xenopus tadpoles reveals a population of T cells that persists through metamorphosis.

To investigate the persistence of larval T lymphocytes in the adult period, tadpoles of the South African clawed frog, Xenopus laevis, were allowed to develop to prometamorphic stages 57-58 and thymectomized (Tx). Thymectomy at this stage allows for maximal expansion of the larval T cell population but prevents emergence of the adult T cell population. Using a T cell-specific monoclonal antibody (mAb) which recognizes the XTLA-1 determinant, we examined the absolute numbers of thymic and splenic T cells expressing XTLA-1 in normal tadpoles, postmetamorphic Tx frogs, and intact age-matched adult frogs. A small, but measurable, number of larvally-derived XTLA-1+ cells persists through metamorphosis. By simultaneously staining with a mAb specific for class II major histocompatibility (MHC) antigens, we determined the phenotype of the persisting XTLA-1+ cells in the Tx frogs. Like XTLA-1+ splenocytes in intact adult controls which are predominantly class II+, most XTLA-1+ cells in Tx adults also express class II. In contrast, most XTLA-1+ cells in the tadpole are class II-. This suggests that a small population of class II+ larval T cells survives metamorphic transition to become a long-lived population in the adult. Alternatively, some class II- larval T cells may express class II in the adult period.

Pronephric carcinoma: chromosomes of cells rescued from apoptosis by an oncogenic herpesvirus detected with a polymerase chain reaction.

The amphibian pronephros is fated to die during early development. Pronephric cells undergo apoptosis and their function is replaced by the mesonephros, which becomes the functional kidney of the adult frog. Tadpoles of the northern leopard frog, Rana pipiens, were inoculated with a Lucké tumour herpesvirus (LTV) preparation. Most of the animals developed typical Lucké renal carcinomas at metamorphosis. Fewer developed carcinomas of the pronephric cell type. A pronephric carcinoma, rescued from apoptosis by the herpesvirus, was harvested from a post-metamorphic frog. The tumour was judged to be pronephric by its anatomical location (in the anterior part of the body) and because both mesonephric kidneys were intact and tumour-free upon removal of the tumour mass. A tumour fragment was fixed for histological examination, which confirmed that the tissue was a renal carcinoma. A further fragment was subjected to short-term culture in order to produce metaphase cells for cytogenetical analysis. Based upon silverstained nucleolar organizing region numbers, 14 of 15 metaphase cells were estimated to have the diploid number (2N = 26) of chromosomes and a karyotype was constructed which did not appear to differ from that of normal cells. A single cell was estimated to be tetraploid (4N = 52). This is the first report of chromosomes of a pronephric Lucké carcinoma. LTV replicates only in tumour tissue maintained in the cold. Because the frog in this study had been maintained in the laboratory at 22 degrees C for about 10 months, no viruses would have been detectable with electron microscopy. However, the presence of Lucké herpesvirus DNA was detected in tumour homogenates by polymerase chain reaction amplification of a 1.2 kbp Hind III restriction fragment of the LTV DNA. The presence of LTV DNA provided assurance that the rescued pronephric tumour was indeed a Lucké carcinoma.

Increased interleukin-6 (IL-6) production in a young child with clinical and pathologic features of multicentric Castleman's disease.

A 21-month-old boy presented with a papular rash, lymphoadenopathy, and splenomegaly. He developed symmetric polyarthritis, fever, and progressive glomerulonephritis. Serologies for viral agents including HIV were negative. Antinuclear antibody was transiently positive, but no anti-DNA antibodies were present. CH50 and serum C3 values were low. Biopsies of skin, kidney, bone marrow, and lymph node were obtained. There was a perivascular and periadnexal lymphocytic infiltrate in the skin, with a normal epidermis. Renal biopsy showed proliferative mesangial glomerulonephritis. Bone marrow showed an increased number of plasma cells. Lymph node showed histologic changes described in multicentric Castleman's disease including marked follicular hyperplasia, vascular proliferation, and interfollicular expansion with numerous plasma cells. IL-6 mRNA was demonstrated in cells in the marginal zone and interfollicular regions of the node by in situ hybridization. Likewise, the serum IL-6 level was elevated during a clinical exacerbation of the patient's nephritis. These data suggest an underlying lymphoproliferative disorder, such as Castleman's disease, with overproduction of IL-6 resulting in systemic features of the disease, including glomerulonephritis.

Fate of herpesvirus DNA in embryos and tadpoles cloned from Lucké renal carcinoma nuclei.

The Lucké renal carcinoma of the northern leopard frog, Rana pipiens, has a herpesvirus aetiology. Lucké tumour nuclei inserted into enucleated frogs' eggs produce development to the swimming tadpole stage. Tissue from these tumour nuclear transplant animals can be induced to survive and differentiate further by allografting to normal tadpoles. We wished to ascertain whether the aetiological agent, the Lucké tumour herpesvirus (LTV), persists in the animals produced by tumour nuclear transplantation. The polymerase chain reaction was used to amplify a 1.2 kbp Hind III restriction fragment of LTV DNA in whole animal homogenates prepared from tumour nuclear transplant tadpoles and normal tadpoles fertilized in vitro. The LTV fragment was not present in the majority (31 of 34) of the cloned animals derived from tumour nuclei, nor was it present in any of five normal tadpoles. Either the 1.2 kbp fragment of LTV DNA was eliminated from most of the cloned animals during the massive reprogramming of the neoplastic genome initiated by insertion of the tumour nuclei into egg cytoplasm, or the nuclei selected for transplantation were primarily those lacking this fragment of LTV DNA. The limited development of the tumour nuclear tadpoles was probably not due to the presence of these viral sequences, but rather reflected the limited plasticity of the tumour cell genome as assayed by nuclear transplantation. Failure to detect the 1.2 kbp fragment of LTV DNA in the majority of mitotic progeny of the Lucké tumour genome does not imply that other parts of the viral genome do not persist.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of DNA sequences of the Lucké herpesvirus in normal and neoplastic kidney tissue of Rana pipiens.

The Lucké tumour herpesvirus (LTV) is the aetiological agent of the Lucké renal adenocarcinoma of the northern leopard frog, Rana pipiens. LTV virions can be detected by electron microscopy in renal adenocarcinomata after prolonged exposure to low temperature. Tumours maintained at warm temperatures do not contain viral particles. To gain insight into the processes of viral infection, replication and oncogenesis, evidence was sought for the presence of LTV DNA in warm renal tumours and normal renal tissue. The polymerase chain reaction was used to amplify a Hind III restriction fragment of LTV DNA in tissue homogenates. LTV DNA was detected in a significant percentage of normal kidneys as well as in "virus-free" warm tumours and virus-containing cold tumours.

The effects of corticosteroid hormones and thyroid hormones on lymphocyte viability and proliferation during development and metamorphosis of Xenopus laevis.

Metamorphosis in the South African clawed frog, Xenopus laevis, is characterized by a striking loss of lymphocytes in the thymus, liver, and spleen. Changes in the proliferative responses of splenocytes and thymocytes to T cell mitogens and semi-allogeneic cells are also observed at metamorphosis. Because the levels of circulating thyroid hormones (TH) and corticosteroid hormones (CH) increase dramatically during the climax of metamorphosis, we have investigated the possible role of TH and CH as mediators of the changes in lymphocyte numbers or lymphocyte function. Here we report on the in vitro effects of CH and TH on lymphocyte viability and on phytohemagglutinin-P (PHA)-stimulated lymphocyte proliferation at prometamorphosis and climax of metamorphosis. We have observed consistently significant inhibition of proliferation by corticosterone. In contrast, we have observed inconsistent inhibition of proliferation by both thyroxine (T4) and triiodothyronine (T3). In short-term studies, the viability of thymocytes and splenocytes was reduced in the presence of CH but not TH. These observations are consistent with a hypothesis that loss of larval lymphocytes and changes of lymphocyte function at metamorphosis may be due to elevated concentrations of CH rather than TH. Because CH have been shown to enhance TH-induced effects during metamorphosis, we looked at the combined effects of these agents on PHA-stimulated lymphocyte proliferation. While each agent was inhibitory in several experiments, there was no significantly greater inhibition when splenic lymphocytes were cultured with both.

Effects of thyroid hormone deprivation on immunity in postmetamorphic frogs.

Previously, we showed that sodium perchlorate treatment of larval frogs (Xenopus laevis) interferes with the normal expansion of T and B lymphocytes and development of an adult-type T-cell population. It was unclear whether these effects resulted from preventing metamorphosis or from long-term thyroid hormone (TH) deprivation. To try to distinguish between these possibilities, we have now studied the effects of perchlorate treatment beginning immediately after metamorphosis. After 5 months, treated animals, but not untreated controls, had large thyroid goiters, were significantly smaller, and had significantly fewer erythrocytes, thymocytes, and splenocytes. Although the number of IgM- splenocytes and thymocytes was reduced, the estimated percent of major histocompatibility complex (MHC) class II+ cells within this subset was not significantly different from that of controls. Furthermore, splenocytes from perchlorate-treated frogs could respond normally by [3H]TdR incorporation to the T-cell mitogens, phytohemagglutinin-P (PHA) and concanavalin A (Con A). Thus, unlike perchlorate-treated larvae, perchlorate-treated juveniles appear to be able to develop T cells with an adult phenotype competent to respond to activation and proliferation signals.