Albumin and a dialyzable serum factor stimulate feeding in vitro by third-stage larvae of the canine hookworm Ancylostoma caninum.

Previous studies demonstrated that third-stage, developmentally arrested larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to canine serum and hostlike temperature. Experiments to determine the identity of the serum stimulus are described. Serum from several nonhost species stimulated feeding, but to levels lower than canine serum. Heating the serum to 57 C had no effect on its stimulatory ability. Dialysis reduced serum stimulatory activity by 50%, and ultrafiltration through 10- and 30-kDa molecular weight cut-off membranes decreased activity in both the filtrates and retentates similarly. Recombination of the filtrates and retentates restored activity to whole serum control levels. Commercial canine and bovine albumin stimulated feeding to serum control levels at 10 and 50 mg/ml, respectively. These results suggest that albumin and an unidentified low molecular weight compound(s) are capable of inducing in vitro feeding by A. caninum L3.     

Host-finding and host recognition of infective Ancylostoma caninum larvae

A. caninum larvae responded to environmental and host stimuli with four behavioral phases of host-finding. (1) Snake-like movement was stimulated by warmth and by defined vibrations of the substratum. (2) Waving behavior was a prerequisite for the passive change-over to dog hairs. It was stimulated by heat radiation and by the CO2 content, warmth, and humidity of an air stream. (3) Creeping direction: the larvae were attracted by heat in temperature gradients as weak as 0.04 degrees C mm-1 and by dog hydrophilic skin surface extracts, but not by skin lipids or serum. (4) Penetration into agar was stimulated by heat, dog hydrophilic skin fraction, and dog serum. The effective component of serum had a molecular weight of between 5000 and 30,000 and proved to be a protein, since it lost its effectiveness after digestion with proteinases. Dog saliva, urine, milk, and various pure dog serum components did not stimulate penetration. A. caninum larvae were able to penetrate mouse skin repeatedly, but they did not follow the tracks of previously penetrated larvae in agar.     

Ac-SAA-1, an immunodominant 16 kDa surface-associated antigen of infective larvae and adults of Ancylostoma caninum

A cDNA encoding a surface-associated antigen was cloned from an Ancylostoma caninum infective larvae (L(3)) cDNA library by immunoscreening with pooled human immune sera. The sera were obtained from individuals living in an Ancylostoma duodenale hookworm-endemic region of China, who had light intensity infections and high antibody titers against A. caninum L(3). Ancylostoma caninum surface-associated antigen-1 is encoded by an 843 bp mRNA with a predicted open reading frame of 162 amino acids. Recombinant Ancylostoma caninum surface-associated antigen-1 was expressed in Escherichia coli and used to prepare a specific antiserum. A Western blot with anti-Ancylostoma caninum surface-associated antigen-1 specific antiserum showed that native Ancylostoma caninum surface-associated antigen-1 protein is expressed by both L(3) and adult hookworms; RT-PCR confirmed that the mRNA is transcribed in both stages. In adult hookworms, the protein localised to the basal layer of the cuticle and hypodermis of adult worms. Serological analysis determined that recombinant Ancylostoma caninum surface-associated antigen-1 protein is recognised by 61% of human sera from a Necator americanus hookworm endemic area in China, indicating the antigen is immunodominant. Anti-Ancylostoma caninum surface-associated antigen-1 antiserum partially inhibited (46.7%) invasion of hookworm L(3) into dog skin in vitro. Together these results suggest that Ancylostoma caninum surface-associated antigen-1 offers promise as a protective vaccine antigen.     

Involvement of Opioid Receptor Subtypes in Both Stimulatory and Inhibitory Effects of the Opioid Peptides on Prolactin Secretion During Pregnancy

1. We have previously demonstrated the existence of a dual neuromodulatory regulation of prolactin secretion by the opioid system. In the present work, we evaluated the opioid receptor subtypes involved in both the stimulatory and the inhibitory regulation of prolactin secretion in pregnant rats. 2. Specific opioid agonists and antagonists were administered intracerebro ventricular (i.c.v.) to rats on day 3 and on day 19 pregnancy in rats of pretreated with mifepristone. Blood samples were obtained after decapitation at 12.00 and 18.00 h. Serum prolactin levels were measured by RIA. 3. The mu-selective agonist DAMGO and beta-endorphin caused a significant increase in serum prolactin secretion on day 3 of pregnancy, during the diurnal surge and intersurge period. Pretreatment with naloxone prevented the increase on prolactin levels induced by DAMGO. The administration of U-50,488, a kappa-selective agonist or DPDPE, a delta-selective agonist, did not modify serum prolactin concentration while the mu1-antagonist naloxonazine reduced significantly serum prolactin levels. On day 19 of pregnancy, the release of prolactin induced by mifepristone was significantly increase by naloxonazine, while the kappa-antagonist nor-binaltorfimine induced only a small but significant increase. No effect was observed after administration of the delta-antagonist naltrindole. 4. We conclude that the mu-opioid receptor seems to be more specifically involved in both the stimulatory and inhibitory regulation by the opioid system on prolactin secretion during pregnancy. The increase on serum prolactin levels on day 3 after administration of DAMGO and beta-endorphin may suggest the participation of other regulatory mechanisms as the dopaminergic and serotoninergic systems. On day 19, only the endogenous ligands delta did not participate in the regulation of prolactin secretion, while the participation of the kappa-opioid receptor was significantly less effective than the endogenous ligand mu. Our results provide evidences of an important role of the opioid system through specific receptors on the regulation of prolactin secretion during early and late pregnancy.     

Phosphoinositide-3-OH-kinase inhibitor LY294002 prevents activation of Ancylostoma caninum and Ancylostoma ceylanicum third-stage infective larvae

The developmentally arrested hookworm infective larva resumes development only after encountering specific host-mediated cues during invasion. These cues activate a signaling pathway that culminates in the resumption of development. In Ancylostoma caninum, activation is characterised by the resumption of feeding and the release of excretory/secretory products required for infection. The dauer stage of the free-living nematode Caenorhabditis elegans is a developmentally arrested stage analogous to the hookworm infective larva. Dauer larvae exit developmental arrest in response to environmental cues that indicate favorable conditions for reproduction and growth. Because of the similarity between dauer recovery and activation, exit from dauer provides a model for hookworm larval activation. An insulin-signaling pathway has been implicated in controlling exit from developmental arrest in both C. elegans dauers and A. caninum larvae. To further investigate the role of insulin signaling in hookworm larval activation, the phosphatidylinositol-3-OH kinase inhibitor LY294002 was tested for its effect on in vitro activation using the resumption of feeding as a marker for activation. LY294002 prevented feeding in A. caninum infective larvae stimulated with host serum filtrate and a glutathione-analogue, the muscarinic agonist arecoline, or the cell permeable cGMP-analogue 8-bromo-cGMP. Similar results were seen with the congeneric hookworm Ancylostoma ceylanicum. These data suggest that an insulin-signaling pathway mediates activation in hookworm larvae, as in C. elegans, and that the phosphatidylinositol-3-OH kinase inhibitor acts downstream of the cGMP and muscarinic signaling steps in the pathway. In A. caninum, LY294002 had no effect on the release of excretory/secretory products associated with activation, suggesting that the secretory pathway diverges from the activation pathway upstream of the phosphatidylinositol-3-OH kinase step. These results provide additional support for the insulin-signaling pathway as the primarily pathway for activation to parasitism in hookworm larvae.     

Ancylostoma caninum: the finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm

Bhopale, V. M., Kupprion, E. K., Ashton, F. T., Boston, R., and Schad, G. A. 2001. Ancylostoma caninum: The finger cell neurons mediate thermotactic behavior by infective larvae of the dog hookworm. Experimental Parasitology 97, 70-76. In the amphids (anteriorly positioned, paired sensilla) of the free-living nematode Caenorhabditis elegans, the so-called finger cells (AFD), a pair of neurons, each of which ends in a cluster of microvilli-like projections, are known to be the primary thermoreceptors. A similar neuron pair in the amphids of the parasitic nematode Haemonchus contortus is also known to be thermoreceptive. The hookworm of dogs, Ancylostoma caninum, has apparent structural homologs of finger cells in its amphids. The neuroanatomy of the amphids of A. caninum and H. contortus is strikingly similar, and the amphidial cell bodies in the lateral ganglia of the latter nematode have been identified and mapped. When the lateral ganglia of first-stage larvae (L1) of A. caninum are examined with differential interference contrast microscopy, positional homologs of the recognized amphidial cell bodies in the lateral ganglia of H. contortus L1 are readily identified in A. caninum. The amphidial neurons in A. caninum were consequently given the same names as those of their apparent homologs in H. contortus. It was hypothesized that the finger cell neurons (AFD) might mediate thermotaxis by the skin-penetrating infective larvae (L3) of A. caninum. Laser microbeam ablation experiments with A. caninum were conducted, using the H. contortus L1 neuronal map as a guide. A. caninum L1 were anesthetized and the paired AFD class neurons were ablated. The larvae were then cultured to L3 and assayed for thermotaxis on a thermal gradient. L3 with ablated AFD-class neuron pairs showed significantly reduced thermotaxis compared to control groups. The thermoreceptive function of the AFD-class neurons associates this neuron pair with the host-finding process of the A. caninum infective larva and shows functional homology with the neurons of class AFD in C. elegans and in H. contortus.     

Response to carbon dioxide by the infective larvae of three species of parasitic nematodes

The response of infective third-stage larvae (L3) of three species of parasitic nematodes, Ancylostoma caninum, Strongyloides stercoralis, and Haemonchus contortus to carbon dioxide (CO(2)) at physiological concentrations was investigated. L3 of the skin-penetrating species, A. caninum and S. stercoralis, were stimulated by CO(2) at the concentration found in human breath (3.3-4%); these larvae responded by crawling actively, but not directionally. Crawling was not stimulated by breath passed through a CO(2)-removing "scrubber" or by "bench air". Both A. caninum and S. stercoralis L3 stopped crawling when exposed to 5% CO(2) for 1 min. L3 of A. caninum became active 9-14 min after exposure to 5% CO(2) ended, but activity resumed more rapidly (10-15 s) if larvae were subsequently exposed to breath or breath through the scrubber. L3 of S. stercoralis resumed crawling 30-35 s after exposure to 5% CO(2), but resumed crawling within a very few seconds when exposed to breath or breath through the scrubber. Thus, while 5% CO(2) was inhibitory, lower concentrations of this gas stimulated L3 of both species. Apparently, exposing immobilized larvae to breath or breath through the scrubber causes the environmental CO(2) concentration to drop to a level that is stimulatory. The L3 of H. contortus ceased crawling and coiled when exposed to human breath or to 1% CO(2), but continued to move within the coil in both cases. The crawling response of the L3 of the two skin-penetrating species, A. caninum and S. stercoralis, to stimulation by CO(2) probably relates to their active host-finding behavior, while the cessation response elicited by CO(2) in H. contortus larvae may relate to the fact that they rely on passive ingestion by a ruminant host.     

人早孕子宫蜕膜催乳素分泌的调节

子宫内膜蜕膜化对胚泡植入与妊娠维持是非常重要的。为探讨蜕膜化维持的调节机制 ,本文研究了妊娠早期人子宫蜕膜细胞催乳素 (PRL)分泌的调节。结果表明 :(1)孕酮显著地刺激PRL的分泌。 (2 )雌激素的作用与其浓度有关 ,生理浓度的雌激素对PRL分泌无明显影响 ,而高水平的雌激素抑制孕酮的刺激作用。合适的雌孕激素比例对蜕膜化的维持是必要的。 (3)RU486明显地抑制PRL的分泌 ,故认为孕酮的作用至少是部分通过受体介导的机制。 (4 )高浓度的cAMP (≥ 10 -5mol/L)显著增加PRL的分泌 ,cAMP信号系统可能在蜕膜化反应中发挥重要作用。     

Role of serum and hormones during the growth and development of rat mammary tumor epithelial cells in collagen gel culture

Summary The characteristics of hormone-dependent rat mammary tumors in response to serum and hormones were determined in collagen gel matrix culture. Epithelial cells from 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary adenocarcinomas were embedded in collagen gel and the effect of estrogen, progesterone, prolactin, insulin, and serum was tested. The total cell number and [³H]thymidine incorporation were used to determine the growth pattern of the cells in culture. It was found that in medium containing 20% porcine serum and supplemented with insulin, estrogen, progesterone and prolactin, both the cell number and [³H]thymidine labeling index increased with time, after an initial lag. Serum seemed to be essential to maintain growth of the tumor cells, because hormones alone, in the absence of serum, were unable to sustain growth of the cells. When estrogen, progesterone, prolactin, and insulin were tested individually in the presence of 20% porcine serum, only estrogen demonstrated a significant stimulatory effect.     

Morning pulse of prolactin maintains seasonal quiescence in the tammar, Macropus eugenii

The pulse of prolactin, present in female tammars kept in an inhibitory photoperiod and absent 5 days after a change to a stimulatory photoperiod, was artificially maintained during stimulatory photoperiods (Exps 1A and 1B) or abolished during an inhibitory photoperiod (Exp. 2). In Exp. 1A, thyrotrophin-releasing hormone (TRH) was injected i.v. for 8 consecutive mornings and in Exp. 1B ovine prolactin was injected i.v. for 10 consecutive mornings beginning on the day the photoperiod was changed from 15L:9D to 12L:12D. In Exp. 1A, in 5 of 6 control females the prolactin pulse was present until the 3rd morning after the lighting was changed, and absent thereafter. In both experiments the early progesterone peak and the interval to birth in the treated females was significantly delayed compared to the control females; reactivation of the CL did not occur until treatment ceased. In Exp. 2, for 5 consecutive mornings, before the prolactin pulse was expected to occur, bromocriptine was injected i.m. In 2/4 treated animals the prolactin pulse was abolished and these animals showed an early progesterone peak and gave birth 28 and 29 days after treatment began. The remaining 2 animals and the controls (N = 4) did not show reactivation until after they experienced a stimulatory photoperiod. The results indicate that the prolactin pulse is the critical factor in the maintenance of the inhibition of the CL of the tammar during seasonal quiescence.     

Hormonal Induction and Roles of Disabled-2 in Lactation and Involution

Disabled-2 (Dab2) is a widely expressed endocytic adaptor that was first isolated as a 96 KDa phospho-protein, p96, involved in MAPK signal transduction. Dab2 expression is lost in several cancer types including breast cancer, and Dab2 is thought to have a tumor suppressor function. In mammary epithelia, Dab2 was induced upon pregnancy and further elevated during lactation. We constructed mutant mice with a mosaic Dab2 gene deletion to bypass early embryonic lethality and to investigate the roles of Dab2 in mammary physiology. Loss of Dab2 had subtle effects on lactation, but Dab2-deficient mammary glands showed a strikingly delayed cell clearance during involution. In primary cultures of mouse mammary epithelial cells, Dab2 proteins were also induced by estrogen, progesterone, and/or prolactin. Dab2 null mammary epithelial cells were refractory to growth suppression induced by TGF-beta. However, Dab2 deletion did not affect Smad2 phosphorylation; rather TGF-beta-stimulated MAPK activation was enhanced in Dab2-deficient cells. We conclude that Dab2 expression is induced by hormones and Dab2 plays a role in modulating TGF-beta signaling to enhance apoptotic clearance of mammary epithelial cells during involution.     

Chemotactic attraction of infective hookworm larvae of Ancylostoma caninum by a dog serum factor

Infective hookworm larvae of Ancylostoma caninum showed chemotaxis on agar plates in a dog serum gradient. This chemotactic behaviour remain unaltered using an ultrafiltrated serum fraction with a molecular weight less than 500. Gelfiltration of this ultrafiltrated fraction revealed a factor with a molecular weight of 480 causing chemotaxis. The chemotactic activity of the factor was destroyed after a pronase treatment. We conclude that the factor could be a polypeptide.     

Resumption of feeding in vitro by hookworm third-stage larvae: a comparative study.

Third-stage infective larvae of the canine hookworm Ancylostoma caninum resume feeding in vitro in response to several stimuli. Experiments were conducted to characterize the in vitro feeding behavior of several hookworm species. Reduced glutathione and, to a lesser extent, canine and human serum stimulated third-stage larvae of Ancylostoma duodenale to resume feeding. Glutathione-induced feeding reached a maximum by 16 hr and was concentration-dependent between 0- and 15-mM glutathione. Oxidized glutathione and the reducing agents dithiothreitol and L-cysteine failed to induce feeding, suggesting that reducing conditions alone were not stimulatory. Serum incubated with glutathione was the most efficient stimulus for Ancylostoma ceylanicum, Ancylostoma braziliense, and Ancylostoma tubaeforme larvae, whereas Uncinaria stenocephala larvae responded best to canine serum alone. Necator americanus larvae did not resume feeding in response to glutathione, serum, glutathione plus serum, or linoleic acid (0.1-10 mM). These differences in feeding behavior suggest that generalizations concerning hookworm biology must be interpreted cautiously.     

Developmental arrest and pregnancy-induced transmammary transmission of Ancylostoma caninum larvae in the murine model

Pregnancy is associated with reactivation of latent infections of many protozoal and helminthic parasites. To facilitate in vivo studies on the process of transmammary transmission of hookworm infection to nursing newborns, we established an experimental model of infection of BALB/c mice with infective larvae of the canine nematode Ancylostoma caninum. To establish latency with a significant reservoir of tissue larvae and achieve acceptable pregnancy success rates, mice were subcutaneously infected at day 5 postimpregnation; similar larval distribution profiles were observed at the end of the gestational period for bred compared to correspondingly infected unbred animals. No larvae were detected in fetuses or neonatal pups. Significant numbers of larvae were not detected in mammary tissue during the periparturient or postpartum lactational periods although about 8% of a dam's reservoir of tissue larvae was transferred to her nursing pups; this suggests that larvae reaching the mammary glands are rapidly transmitted through the milk sinuses, as was documented by histopathological analyses. Comparison of BALB/c with C57BL/6 mice that typically display divergent immune responses to infection showed no difference in tissue larval burden or in numbers transferred to pups. A hypothesis for the molecular mechanism of larval reactivation and transmission is discussed.     

The positive regulatory effect of TGF-beta2 on primitive murine hemopoietic stem and progenitor cells is dependent on age, genetic background, and serum factors

TGF-beta is considered a negative regulator of hemopoietic stem and progenitor cells. We have previously shown that one TGF-beta isoform, TGF-beta2, is, in fact, a positive regulator of murine hemopoietic stem cell function in vivo. In vitro, TGF-beta2, but not TGF-beta1 and TGF-beta3, had a biphasic dose response on the proliferation of purified lin-Sca1(++)kit(+) (LSK) cells, with a stimulatory effect at low concentrations, which was subject to mouse strain-dependent variation. In this study we report that the stimulatory effect of TGF-beta2 on the proliferation of LSK cells increases with age and after replicative stress in C57BL/6, but not in DBA/2, mice. The age-related changes in the TGF-beta2 effect correlated with life span in BXD recombinant strains. The stimulatory effect of TGF-beta2 on the proliferation of LSK cells requires one or more nonprotein, low m.w. factors present in fetal calf and mouse sera. The activity of this factor(s) in mouse serum increases with age. Taken together, our data suggest a role for TGF-beta2 and as yet unknown serum factors in the aging of the hemopoietic stem cell compartment and possibly in organismal aging.     

Receptors and neurotransmitters involved in the dual modulation of prolactin release by the serotoninergic system in pregnant and lactating rats

The receptors and neurotransmitter pathways that may participate in the inhibitory action of 5-hydroxytryptamine (5HT) on prolactin release during late pregnancy and lactation in rats were studied. Administration of the 5HT synthesis inhibitor, p-chlorophenylalanine, to late pregnant rats induced a significant increase in serum prolactin concentrations at 17:00 h on day 19 of pregnancy that was partially blocked by injections of the 5HT precursor, 5-hydroxytryptophan, or the 5HT agonists, 8-hydroxy-2-(di-n-propylamino)-tetralin hydrobromide (S1a), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (S2) and N-(3-chlorophenyl)imidodicarbonimide diamide HCl (S3), but not by RU 24969 (S1b) or 1-meta-(chlorophenyl)-piperazine-2-HCl (S1a-2c). The 5HT neurotoxins, fenfluramine and p-chloroamphetamine, which selectively destroy fine axon serotoninergic fibres but not coarse ones, prevented the increase in circulating prolactin observed at 18:00 h on pro-oestrus and on day 21 of pregnancy, but did not modify serum prolactin concentrations at 17:00 h on day 19 of pregnancy. Administration of the adrenergic antagonists, metoprolol or prazosin, also prevented the stimulatory effects of p-chlorophenylalanine or ketanserin in pregnant rats on day 19 (17:00 h) or on days 10-12 (16:30 h) in lactating rats separated from their litters. Administration of p-chlorophenylalanine to pregnant rats on day 19 reduced dopamine concentrations in the arcuate nucleus and in the anterior hypothalamus and noradrenaline concentrations in the anterior hypothalamus and the suprachiasmatic nucleus. These results indicate that the inhibitory actions of 5HT on prolactin release in pregnant and lactating rats are mediated by S1a, S2a and S3 receptors and by the coarse axon serotoninergic fibres. In addition, the inhibitory actions of 5HT may modulate the action of a stimulatory adrenergic pathway, as well as the concentrations of noradrenaline and dopamine in different hypothalamic areas, which, in turn, particularly arcuate nucleus dopamine, regulate prolactin release.     

Proceedings: Neurohormonal control of prolactin release

Neurohomonal control of prolactin release was studied in pseudopregnant and pregnant rats. Nembutal administered at 1300 hours on Day 3 of pseudopregnancy prevented prolactin release which normally occurred at 1700 hours of the same day. Antiestrogen administered the day before did not prevent prolactin release but ovariectomy did. Estrogen administered immediately after ovariectomy did not restore prolactin secretion; however, progesterone on Day 3 in the ovariectomized-estrogen treated induced an increase in prolactin at 1700 hours. Progesterone was capable of increasing prolactin release the first 5 days of pseudopregnancy but not Days 6-12 when prolactin values were low. A similar effect was seen the first 7 days of pregnancy. Progesterone, but not estrogen, modified prolactin values on Day 9 at 1700 hours. Ovariectomy on Day 19 of pregnancy induced prolactin release within 4 hours and persisted for 58 hours. Progesterone administration immediately after ovariectomy prevented prolactin release for a few hours. These results suggest that the regulation of prolactin release by the central nervous system depends on the circulating estrogen/progesterone ratio, since estrogen facilitated prolactin release when plasma progesterone was low and progesterone induced prolactin release when adequated levels of estrogen existed, but exerted an inhibitory action when estrogen was not present.     

Pneumonyssoides caninum: a mite from the nasal cavities and frontal sinuses of the dog. A case report

The dog nasal mite, Pneumonyssoides caninum, is reported from Italy for the first time. The identification of the mite was based upon the morphology of larvae and adults.     

Protection against vertical transmission in bovine neosporosis

In this study we were interested to determine whether infection of cattle prior to pregnancy would afford any protection to the foetus if the dams were challenged with Neospora caninum at mid-gestation. The experiment comprised four groups of cattle: group 1, uninfected controls; group 2, inoculated with N. caninum tachyzoites 6 weeks prior to mating and then challenged with N. caninum at mid-gestation; group 3, naive cattle challenged with N. caninum at mid-gestation and group 4 were infected with N. caninum prior to mating and left unchallenged throughout pregnancy. Positive cell-mediated and humoral immune responses to N. caninum were recorded in groups 2 and 4 prior to pregnancy and in groups 2, 3 and 4 following challenge at mid-gestation. However there was a marked down regulation of the cell-mediated immune response in all groups around mid-gestation. There was a significant increase in rectal temperature response in animals in group 3 compared to group 2 following challenge but no other clinical symptoms of disease were recorded and all cattle proceeded to calving. At calving, pre-colostral blood samples were negative for antibodies to N. caninum in all the calves born to dams in groups 1, 2 and 4. In contrast, all the calves born to dams in group 3 had high levels of specific antibody to N. caninum indicating that they had been exposed to the parasite in utero. At post-mortem N. caninum DNA was detected in CNS, thymus and placental cotyledon samples in calves from group 3. All tissue samples from calves in the other 3 groups were negative for N. caninum DNA with the exception of one calf from group 2 where specific DNA was detected in a sample of spinal cord. These results suggest that the immune response generated in the dams in group 2 prior to pregnancy had protected against vertical transmission of the parasite following challenge at mid-gestation.