Investment of both essential fatty and amino acids to immunity varies depending on reproductive stage.

Trade-offs among the key life-history traits of reproduction and immunity have been widely documented. However, the currency in use is not well-understood. We investigated how reproducing female side-blotched lizards, Uta stansburiana, allocate lipids versus proteins when given an immune challenge. We tested whether lizards would invest more in reproduction or immunity depending on reproductive stage. Females were given stable isotopes (15 N-leucine and 13 C-1-palmitic acid), maintained on a regular diet and given either a cutaneous biopsy or a sham biopsy (control). Stable isotopes were monitored and analyzed in feces and uric acid, skin biopsies, eggs, and toe clips. We found that lizards deposited both proteins and lipids into their healing wounds (immune-challenged), skin (control), and eggs (all) and that catabolism of proteins exceeded incorporation into tissue during wound-healing. Specifically, we found that healed biopsies of wounded animals had more leucine and palmitic acid than the nonregrown skin biopsies taken from unwounded control animals. Earlier in reproduction, lizards invested relatively more labeled proteins into healing their wound tissue, but not into unwounded skin of control animals. Thus, reproduction is sometimes favored over self-maintenance, but only in later reproductive stages. Finally, we documented positive relationships among the amount of palmitic acid deposited in the eggs, the amount of food eaten, and the amount of palmitic acid excreted, suggesting higher turnover rates of lipids in lizards investing highly in their eggs.

Metabolic responses to different immune challenges and varying resource availability in the side-blotched lizard (Uta stansburiana).

The energetic cost of immunity depends on many factors, including the type of challenge, the timing of the response, and the state of the animal. We measured changes in the standard metabolic rates of side-blotched lizards (Uta stansburiana Baird and Girard, 1852) in response to different immune challenges and nutritional states. In the first experiment, lizards were randomly assigned to one of four treatments: lipopolysaccharide (LPS) injection (to stimulate the response to a pathogen), cutaneous biopsy (as a proxy to a superficial wound), both injection and biopsy, or neither (control). Four and five days later, we measured the standard metabolic rates of the lizards. In response to healing a cutaneous wound, lizards reduced metabolic rate and lost body mass. Healing rate was also inversely related to weight loss, but LPS had no effect on body mass or metabolic rate. In the second experiment, a new set of lizards were randomly assigned to a high-food or low-food diet and administered a cutaneous biopsy. As in the first experiment, we observed a reduction in metabolic rate after wounding; moreover, this decrease was positively correlated with the rate of healing. We observed higher rates of metabolism in lizards that ate more food, but food consumption was unrelated to the decrease in metabolic rate following the biopsy. These experiments demonstrate the dynamic nature of the immune response in response to immune challenge and the state of the organism.

Encapsulation of the Acanthocephalan Sphaerirostris picae in Its Natural Paratenic Host, Lacerta agilis.

This study presents the structure of the capsule around the acanthocephalan Sphaerirostris picae (Rudolphi, 1819) Golvan, 1956, in its natural paratenic host, Lacerta agilis Linnaeus, 1758. The capsule was composed of 2 layers: a thin, dense inner layer and a loose, thick outer layer. The inner layer was formed by macrophages and multinucleated cells. The outer layer consisted mainly of flattened fibroblasts, which included a small number of macrophages, granulocytes, plasma cells, and pigment cells; the extensive intercellular spaces between these cells were filled with collagen fibers. The acanthocephalans are thought to have particular defense mechanisms that diminish phagocytic activity of immune system cells in the host.

Immune responses of a wall lizard to whole-body exposure to radiofrequency electromagnetic radiation.

PURPOSE: During the last three decades, the number of devices that emit non-ionizing electromagnetic radiation (EMR) at the wireless communication spectrum has rapidly increased and possible effects on living organisms have become a major concern. The purpose of this study was to investigate the effects of radiofrequency EMR emitted by a widely used wireless communication device, namely the Digital Enhanced Communication Telephony (DECT) base, on the immune responses of the Aegean wall lizard (Podarcis erhardii). MATERIALS AND METHODS: Adult male lizards were exposed 24 h/day for 8 weeks to 1880-1900 MHz DECT base radiation at average electric field intensity of 3.2 V/m. Immune reactivity was assessed using the phytohemagglutinin (PHA) skin swelling and mixed lymphocyte reaction (MLR) tests. RESULTS: Our results revealed a noticeable suppression (approximately 45%) of inflammatory responses in EMR-exposed lizards compared to sham-exposed animals. T cell-mediated responses were marginally affected. CONCLUSION: Daily radiofrequency EMR exposure seems to affect, at least partially, the immunocompetence of the Aegean wall lizard.

CD59 mediates cartilage patterning during spontaneous tail regeneration.

The regeneration-competent adult animals have ability to regenerate their lost complex appendages with a near-perfect replica, owing to the positional identity acquired by the progenitor cells in the blastema, i.e. the blastemal cells. CD59, a CD59/Ly6 family member, has been identified as a regulator of positional identity in the tail blastemal cells of Gekko japonicus. To determine whether this function of CD59 is unique to the regenerative amniote(s) and how CD59 mediates PD axis patterning during tail regeneration, we examined its protective role on the complement-mediated cell lysis and intervened CD59 expression in the tail blastemal cells using an in vivo model of adenovirus transfection. Our data revealed that gecko CD59 was able to inhibit complement-mediated cell lysis. Meanwhile, CD59 functioned on positional identity through expression in cartilage precursor cells. Intervening positional identity by overexpression or siRNA knockdown of CD59 resulted in abnormal cartilaginous cone patterning due to the decreased differentiation of blastemal cells to cartilage precursor cells. The cartilage formation-related genes were found to be under the regulation of CD59. These results indicate that CD59, an evolutionarily transitional molecule linking immune and regenerative regulation, affects tail regeneration by mediating cartilage patterning.

Granulocytes of reptilian sauropsids contain beta-defensin-like peptides: a comparative ultrastructural survey.

The ability of lizards to withstand infections after wounding or amputation of the tail or limbs has suggested the presence of antimicrobial peptides in their tissues. Previous studies on the lizard Anolis carolinensis have identified several beta-defensin-like peptides that may potentially be involved in protection from infections. The present ultrastructural immunocytochemical study has analyzed tissues in different reptilian species in order to localize the cellular source of one of the more expressed beta-defensins previously sequenced in lizard indicated as AcBD15. Beta-defensin-like immunoreactivity is present in some of the larger, nonspecific granules of granulocytes in two lizard species, a snake, the tuatara, and a turtle. The ultrastructural study indicates that only heterophilic and basophilic granulocytes contain this defensin while other cell types from the epidermis, mesenchyme, and dermis, muscles, nerves, cartilage or bone are immunonegative. The study further indicates that not all granules in reptilian granulocytes contain the beta-defensin peptide, suggesting the presence of granules with different content as previously indicated for mammalian neutrophilic leucocytes. No immunolabeling was instead observed in granulocytes of the alligator and chick using this antibody. The present immunocytochemical observations suggest a broad cross-reactivity and conservation of beta-defensin-like sequence or steric motif across lepidosaurians and likely in turtles while archosaurian granulocytes may contain different beta-defensin-like or other peptides.

Immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis: an in vitro study.

Present in vitro investigation for the first time in ectotherms demonstrated the immunomodulatory role of substance P in the wall lizard Hemidactylus flaviviridis. Substance P inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes. Inhibitory effect of substance P was completely blocked by NK-1 receptor antagonist spantide I, indicating the NK-1 receptor mediated action. Further, NK-1 receptor-coupled downstream signaling cascade involved in controlling phagocytosis was explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both the inhibitors, in a concentration-related manner decreased the suppressive effect of substance P on phagocytosis. In addition, substance P treatment caused an increase in intracellular cAMP level in splenic phagocytes. Taken together, it can be suggested that substance P via NK-1 receptor-coupled AC-cAMP-PKA pathway modulated the phagocytic activity of splenic phagocytes in wall lizards.

Role of gonadotropin and Leydig cell-secreted factors in the control of testicular macrophage activities in the wall lizard Hemidactylus flaviviridis.

The present in vitro study for the first time demonstrates the endocrine and paracrine control of testicular macrophage activities in ectodermic vertebrates. Follicle-stimulating hormone (FSH) increased phagocytosis and superoxide production by macrophages. In regard to paracrine control, non-activated Leydig cell-conditioned medium (LCCM) decreased both the activities, whereas FSH-preactivated LCCM had differential effects: inhibitory on phagocytosis and stimulatory on superoxide production. However, FSH-activated LCCM, in addition to superoxide production, also enhanced phagocytosis. After heat inactivation, FSH-activated LCCM inhibited both the activities. Addition of FSH resulted in stimulation of phagocytosis, while partially restored the superoxide production. It can be speculated that androgen in heat-inactivated FSH-activated LCCM, in the presence of FSH, instead of inhibitory had stimulatory effect on phagocytosis, but remained inhibitory to superoxide production. Further, FSH-induced Leydig cell-secreted non-steroidal heat-labile factors appear to have stimulatory effect on superoxide production. This was corroborated by experiments with dihydrotestosterone in presence/absence of FSH.

Immune function varies with reproductive stage and context in female and male tree lizards, Urosaurus ornatus.

Competition among physiological processes for limited resources often results in trade-offs. Key among these processes is reproduction and immune function, and optimizing both appears to be difficult. To test the hypothesis that the resource demands of reproduction compromise immune function, we measured rates of wound healing, an integrated measure of innate immunity, across different reproductive stages in female and male tree lizards (Urosaurus ornatus) in both the field and the laboratory. The hypothesis predicted that immune function would be lowest when resource demands of reproduction are highest, i.e., vitellogenic females and reproductive males. In the field, vitellogenic females had significantly slower healing rate than females in other reproductive stages. However, in the laboratory, vitellogenic females had a healing rate similar to that of other females. Conversely, males showed suppression of healing in the laboratory but not in the field during the reproductive season. The results of this study support a trade-off between reproduction and immune function, and suggest that reproduction is given priority. However, the results also indicate that this trade-off is not fixed in the reproductive process and that it may instead be dependent on the context, sex or resource balance of the individual.

Endocrine roles of D-aspartic acid in the testis of lizard Podarcis s. sicula.

In the lizard Podarcis s. sicula, a substantial amount of D-aspartate (D-Asp) is endogenous to the testis and shows cyclic changes of activity connected with sex hormone profiles during the annual reproductive phases. Testicular D-Asp content shows a direct correlation with testosterone titres and a reverse correlation with 17beta-estradiol titres. In vivo experiments, consisting of i.p. injections of 2.0 micromol/g body weight of D-Asp or other amino acids, in lizards collected during the three main phases of the reproductive cycle (pre-reproductive, reproductive and post-reproductive period), revealed that the testis can specifically take up and accumulate D-Asp alone. Moreover, this amino acid influences the synthesis of testosterone and 17beta-estradiol in all phases of the cycle. This phenomenon is particularly evident during the pre- and post-reproductive period, when endogenous testosterone levels observed in both testis and plasma were the lowest and 17beta-estradiol concentrations were the highest. D-Asp rapidly induces a fall in 17beta-estradiol and a rise in testosterone at 3 h post-injection in the testis and at 6 h post-injection in the blood. In vitro experiments show that testicular tissue converted L-Asp into D-Asp through an aspartate racemase. D-Asp synthesis was measured in all phases of the cycle, but was significantly higher during the reproductive period with a peak at pH 6.0. The exogenous D-Asp also induces a significant increase in the mitotic activity of the testis at 3 h (P < 0.05) and at 6 h (P < 0.01). Induction of spermatogenesis by D-Asp is recognized by an intense immunoreactivity of the germinal epithelium (spermatogonia and spermatids) for proliferation cell nuclear antigen (PCNA). The effects of D-Asp on the testis appear to be specific since they were not seen in lizards injected with other D- or L-forms of amino acids with known excitatory effects on neurosecretion. Our results suggest a regulatory role for D-Asp in the steroido-genesis and spermatogenesis of the testis of the lizard Podarcis s. sicula.

Dual mode of catecholamine action on splenic macrophage phagocytosis in wall lizard, Hemidactylus flaviviridis.

In the present study, in vitro concentration-related effect of catecholamines, dopamine (DA), norepinephrine (NE), and epinephrine (E) was observed on phagocytic activity of splenic macrophages to understand the impact of sympatho-adrenal-medullary (SAM) activation on innate immunity in wall lizard Hemidactylus flaviviridis under stress condition. Restraint stress for 1 h resulted in marked suppression of macrophage phagocytosis, suggesting that supra-physiological level of catecholamines in response to SAM activation under stress suppressed phagocytosis. This interpretation was reinforced since all the catecholamines considerably reduced phagocytosis at high concentrations ranging from 10(-7) to 10(-5)M. On the contrary, DA, NE, and E at low concentrations considerably stimulated phagocytosis, which increased with the decrease of concentrations ranging from 10(-11) to 10(-15)M. Further, effect of NE and E was blocked by beta-adrenergic blocker suggesting the beta-adrenoceptor-dependent regulating mechanism of NE and E. DA acts through both beta-adrenoceptor-dependent and D1/D2 class receptor-dependent mechanism, since beta-adrenergic blocker could partially block the DA effect. beta-Adrenoceptor-linked adenylate cyclase-mediated cAMP action in modulation of phagocytic activity was evident as 3-isobutyl-1-methyl-xanthine suppressed phagocytosis. Further, to delineate the mode of dual effect of catecholamines through beta-adrenergic receptors, in vitro concentration-related effect of cAMP was investigated on macrophage phagocytosis. cAMP depending on concentration had opposite effect on phagocytosis, and its stimulatory effect at low concentrations was reversed by actinomycin D and cycloheximide, whereas these transcription and translation inhibitors, respectively, failed to alter the inhibitory effect of cAMP at high concentrations. This suggests the concentration-related two different pathways of catecholamine action, classical non-genomic at high concentration while genomic pathway at low concentration.

In vitro effect of sex steroids on cytotoxic activity of splenic macrophages in wall lizard (Hemidactylus flaviviridis).

Sexual dimorphism was observed in nitrite release and IL-1-like molecule production by splenic macrophages of the wall lizard (Hemidactylus flaviviridis), with a higher level in females than in males. Gonadectomy in both males and females resulted in a considerable increase of nitrite and IL-1-like molecule secretion, suggesting that the sex hormones inhibit cytotoxic activity of macrophages. To verify this assumption, dose- and time-related in vitro experiments with male and female sex steroids, dihydrotestosterone (DHT) and 17beta-estradiol (E(2)), respectively, were carried out. E(2) and DHT both significantly reduced the nitrite release and IL-1-like molecule production with an increase of dose or duration of treatment.

Resistance of a lizard (the green anole, Anolis carolinensis; Polychridae) to ultraviolet radiation-induced immunosuppression.

The green anole (Anolis carolinensis) is the most northerly distributed of its Neotropical genus. This lizard avoids a winter hibernation phase by the use of sun basking behaviors. Inevitably, this species is exposed to high doses of ambient solar ultraviolet radiation (UVR). Increases in terrestrial ultraviolet-B (UV-B) radiation secondary to stratospheric ozone depletion and habitat perturbation potentially place this species at risk of UVR-induced immunosuppression. Daily exposure to subinflammatory UVR (8 kJ/m2/day UV-B, 85 kJ/m2/day ultraviolet A [UV-A]), 6 days per week for 4 weeks (total cumulative doses of 192 kJ/m2 UV-B, 2.04 x 10(3) kJ/m2 UV-A) did not suppress the anole's acute or delayed type hypersensitivity (DTH) response to horseshoe crab hemocyanin. In comparison with the available literature UV-B doses as low as 0.1 and 15.9 kJ/m2 induced suppression of DTH responses in mice and humans, respectively. Exposure of anoles to UVR did not result in the inhibition of ex vivo splenocyte phagocytosis of fluorescein labeled Escherichia coli or ex vivo splenocyte nitric oxide production. Doses of UV-B ranging from 0.35 to 45 kJ/m2 have been reported to suppress murine splenic/peritoneal macrophage phagocytosis and nitric oxide production. These preliminary studies demonstrate the resistance of green anoles to UVR-induced immunosuppression. Methanol extracts of anole skin contained two peaks in the ultraviolet wavelength range that could be indicative of photoprotective substances. However, the resistance of green anoles to UVR is probably not completely attributable to absorption by UVR photoprotective substances in the skin but more likely results from a combination of other factors including absorption by the cutis and absorption and reflectance by various components of the dermis.

Helodermin- and helospectin-like immunoreactivities in the rat brain: an immunochemical and immunohistochemical study.

Helodermin is an amidated peptide of 35 amino acid residues isolated from the lizard Heloderma suspectum. Homologous peptides, helospectins I and II, peptides of 38 and 37 amino acid residues, respectively, have been isolated from the lizard Heloderma horridum. This group of peptides stimulates the adenylate cyclase activity. Helodermin- and helospectin-like immunoreactivities were studied in the rat brain by using immunohistochemistry and radioimmunoassay in combination with high-performance liquid chromatography. The highest concentrations of helodermin-like immunoreactivity were found in the cerebellum and hypothalamus. The chromatographic analysis of rat brain extract revealed one main immunoreactive peak with elution properties similar to those of authentic lizard helodermin. Helodermin-immunoreactive neurons were located in the supraoptic nucleus, suprachiasmatic nucleus, periventricular nucleus, arcuate nucleus and central gray. Fibers and terminals of varying densities were observed in the bed nucleus of the stria terminalis, medial part of the central nucleus of amygdala, external layer of the median eminence, thalamus and central gray. The highest concentrations of helospectin-like immunoreactivity were found in the cerebral cortex, hypothalamus and medulla. The chromatographic analysis of brain extract revealed one major peak with elution properties similar to those of authentic helospectin I. Helospectin-immunoreactive neurons were located in the suprachiasmatic nucleus, central gray, cerebral cortex, dorsomedial hypothalamic nucleus and supramammillary nucleus. Helospectin-immunoreactive fibers and terminals were found in the bed nucleus of the stria terminalis, medial part of the central nucleus of amygdala, median eminence, lateral parabrachial nucleus, central gray, cerebral cortex, thalamus and nucleus of the solitary tract. The present study has revealed novel neuronal systems in the rat brain by using antisera against the lizard peptides helodermin and helospectin. The patterns of immunostaining suggest a role for the helodermin- and helospectin-like peptides in the hypothalamo-hypophyseal control of endocrine functions.

Neuron-specific monoclonal antibodies raised against the low molecular weight fraction of a brain homogenate of the pond snail Lymnaea stagnalis immunoreact with neurons in the central nervous system of the cockroach, the guppy, the wall lizard, the rat and man.

Monoclonal antibodies were raised against the small molecular weight fraction (less than 30 kilodaltons) of an extract from 200 central nervous systems (CNS) of the freshwater snail Lymnaea stagnalis. In a first screening step the supernatants of the 297 emerging hybridomas were immunocytochemically tested on sections of the CNS of L. stagnalis. Sixty-six appeared to produce neuron-specific antibodies, five reacted with non-neuronal elements. In a second step the 66 neuron-specific antibodies were tested on sections of the CNS of the guppy. Three reacted positively. In the third step the three antibodies were tested on the CNS of the rat. One antibody (Mab4H5) appeared to give positive results. In the snail brain Mab4H5 stains two identified giant neurons, one in the visceral ganglion (VD1), and one in the right parietal ganglion (RPD2)--these neurons form part of the network controlling the respiratory system--and a small number of cells in the cerebral ganglia (in the anterior and ventral lobes). Ultrastructural observations using immunogold labelling in VD1 showed the antigen to be localized to the secretory vesicles. In the guppy Mab4H5 stains fibres in the tectum and cell bodies in the reticular formation. In rat CNS staining was observed in Purkinje neurons of the cerebellum, in cortical pyramidal neurons and in neurons and fibres in other brain areas. Subsequent Mab4H5 staining of the CNS of the lizard, the cockroach and parts of the human CNS showed that these tissues also contain Mab4H5-positive neurons. In the human cortex and cerebellum the staining pattern appeared to be similar to that of the rat. On the basis of the results it is hypothesized that the antibody reacts with phylogenetically ancient amino acid sequences.

Immunological changes during pregnancy in the viviparous lizard, Chalcides ocellatus.

Splenic cells from pregnant and non-pregnant viviparous lizards (Chalcides ocellatus) were stimulated in vitro with the mitogens, concanavalin A (Con A), phytohemagglutinin (PHA) and Escherichia coli lipopolysaccharide (LPS). Cell cultures from pregnant animals were significantly less responsive to Con A and PHA than comparable cultures from non-pregnant animals. The response was depressed during the first period of pregnancy and remained low in magnitude until parturition. By contrast, the response of maternal splenic cells to LPS was reduced in pregnant lizards only during advanced pregnancy. The drastic decrease in mitogenic responsiveness was associated with marked involution of the maternal spleen. These findings strongly suggest that pregnancy impairs the immunoreactivity of viviparous lizards. Possible mechanisms for this impairment and the relationship to circulating levels of sex hormones are discussed.

Endogenous corticosteroids mediate seasonal cyclic changes in immunity of lizards.

Endogenous corticosteroid (CS) blood levels were radioimmunoassayed in fresh, field-collected lizards Chalcides ocellatus at two week-intervals throughout the four consecutive seasons. These animals were used in parallel to investigate the splenic T and B lymphocyte level, lymphoproliferative responsiveness to concanavalin A and primary antibody production in vitro against rat erythrocytes (RRBC). The recorded data indicated that fully developed splenic lymphoid tissue and powerful immune responsiveness are coincident with a continuously low CS level, and characterize the period from spring through early autumn. On the other hand, the dramatic lymphocytic destruction and impairment of immune reactivity observed in autumn and winter are associated with not only a high, but above all sustained, rise in endogenous CS levels. Apparently, exposure of lizard lymphocytes to comparatively high, yet physiologic, levels of endogenous CS for prolonged periods of time lead to impairment of their immune functions. In support, long-term administration of exogenous hydrocortisone acetate (HC) to "summer" lizards resulted in a high and lasting elevation in blood CS levels that was associated with a considerable depletion of lymphoid elements and abrogation of immune reactivity, exactly as in normal lizards collected from the field in autumn through winter. In addition, pharmacologic inhibition of CS synthesis by administration of metyrapone at the beginning of autumn greatly modulated the lizard lymphocyte response to the autumn-related immunodepression. The study thus strongly suggests that the autumn/winter-dependent immunosuppression in lizards is essentially due to a high and lasting rise in levels of endogenous CS. The results are discussed from the perspective of the role played by CS in mediating the seasonal rhythms that affect reptilian immunity.

Characteristics of heterologous anti lizard (Calotes versicolor) thymocyte serum which identify the functions of thymus-derived cell lineage.

In an attempt to study the functions of thymus derived cell lineage, two different heterologous anti-lizard thymocyte sera (ATS i.v. & ATS i.p) were raised by immunizing rabbits with viable thymocytes of Calotes versicolor. Cytotoxicity assay, nylon wool fractionation and thymectomy were employed to assess the effect of these ATS on lymphoid cells of the lizards. The results revealed that ATS i.v. contained minimum amount of irrelevant antibodies than ATS i.p; non-adherent splenocytes were enriched with ATS sensitive cells, whereas the adherent with SIg+; adult thymectomy decreased ATS sensitive cells in spleens and the density of thymic antigen (identified by the ATS) in splenic thymus derived cell lineage was much higher than on thymocytes themselves. The cells of thymic lineage identified by the ATS thus resemble those of higher vertebrates.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. I. Response of lymphoid tissues and cells to in vivo and in vitro hydrocortisone.

A single dose of 1 mg/g body weight of hydrocortisone acetate (HC) administered intraperitoneally to adult lizards, Chalcides ocellatus induced rapidly a reduction of about 85% of thymic lymphocytes. Histological evidence indicated that cortical, as well as, medullary thymocytes are sensitive to HC exposure. Around 40-50% of lymphocytes in peripheral blood (PB) and spleen were depleted at 3-7 days post-HC injection; such depletion durated about 4 weeks for PB but was rather temporary in spleen. Increase in number of bone marrow (BM) lymphocytes was negligible and transient and could by no way account for the dramatic cell losses in the different lymphoid tissues. The findings thus suggested that HC-mediated lymphocyte depletion in lizards is not attributable to redistribution between the different lymphoid compartments but rather to destruction. In direct conformation, lymphocytes were readily lysed in vitro by 10(-3)M HC, thymocytes being more vulnerable greater than PB greater than spleen greater than BM lymphocytes.