Clodronate Liposome-Mediated Phagocytic Hemocyte Depletion Affects the Regeneration of the Cephalic Tentacle of the Invasive Snail, Pomacea canaliculata.

After amputation, granular hemocytes infiltrate the blastema of regenerating cephalic tentacles of the freshwater snail Pomacea canaliculata. Here, the circulating phagocytic hemocytes were chemically depleted by injecting the snails with clodronate liposomes, and the effects on the cephalic tentacle regeneration onset and on Pc-Hemocyanin, Pc-transglutaminase (Pc-TG) and Pc-Allograft Inflammatory Factor-1 (Pc-AIF-1) gene expressions were investigated. Flow cytometry analysis demonstrated that clodronate liposomes targeted large circulating hemocytes, resulting in a transient decrease in their number. Corresponding with the phagocyte depletion, tentacle regeneration onset was halted, and it resumed at the expected pace when clodronate liposome effects were no longer visible. In addition to the regeneration progress, the expressions of Pc-Hemocyanin, Pc-TG, and Pc-AIF-1, which are markers of hemocyte-mediated functions like oxygen transport and immunity, clotting, and inflammation, were modified. After the injection of clodronate liposomes, a specific computer-assisted image analysis protocol still evidenced the presence of granular hemocytes in the tentacle blastema. This is consistent with reports indicating the large and agranular hemocyte population as the most represented among the professional phagocytes of P. canaliculata and with the hypothesis that different hemocyte morphologies could exert diverse biological functions, as it has been observed in other invertebrates.

The Eco-Immunological Relevance of the Anti-Oxidant Response in Invasive Molluscs.

Reactive oxygen species (ROS) are volatile and short-lived molecules playing important roles in several physiological functions, including immunity and physiological adaptation to unsuitable environmental conditions. In an eco-immunological view, the energetic costs associated with an advantageous metabolic apparatus able to cope with wide changes in environmental parameters, e.g., temperature range, water salinity or drought, could be further balanced by the advantages that this apparatus may also represent in other situations, e.g., during the immune response. This review provides an overview of molluscs included in the IUCN list of the worst invasive species, highlighting how their relevant capacity to manage ROS production during physiologically challenging situations can also be advantageously employed during the immune response. Current evidence suggests that a relevant capacity to buffer ROS action and their damaging consequences is advantageous in the face of both environmental and immunological challenges, and this may represent a trait for potential invasiveness. This should be considered in order to obtain or update information when investigating the potential of the invasiveness of emerging alien species, and also in view of ongoing climate changes.

The complex relationship between female age and embryo euploidy.

BACKGROUND: Female age is the strongest predictor of embryo chromosomal abnormalities and has a nonlinear relationship with the blastocyst euploidy rate: with advancing age there is an acceleration in the reduction of blastocyst euploidy. Aneuploidy was found to significantly increase with maternal age from 30% in embryos from young women to 70% in women older than 40 years old. The association seems mainly due to chromosomal abnormalities occurring in the oocyte. We aimed to elaborate a model for the blastocyst euploid rate for patients undergoing in-vitro fertilization/intra cytoplasmic sperm injection (IVF/ICSI) cycles using advanced machine learning techniques. METHODS: This was a retrospective analysis of IVF/ICSI cycles performed from 2014 to 2016. In total, data of 3879 blastocysts were collected for the analysis. Patients underwent PGT-Aneuploidy analysis (PGT-A) at the Center for Reproductive Medicine of European Hospital (Rome, Italy) have been included in the analysis. The method involved whole-genome amplification followed by array comparative genome hybridization. To model the rate of euploid blastocysts, the data were split into a train set (used to fit and calibrate the models) and a test set (used to assess models' predictive performance). Three different models were calibrated: a classical linear regression; a gradient boosted tree (GBT) machine learning model; a model belonging to the generalized additive models (GAM). RESULTS: The present study confirms that female age, which is the strongest predictor of embryo chromosomal abnormalities, and blastocyst euploidy rate have a nonlinear relationship, well depicted by the GBT and the GAM models. According to this model, the rate of reduction in the percentage of euploid blastocysts increases with age: the yearly relative variation is -10% at the age of 37 and -30% at the age of 45. Other factors including male age, female and male Body Mass Index, fertilization rate and ovarian reserve may only marginally impact on embryo euploidy rate. CONCLUSIONS: Female age is the strongest predictor of embryo chromosomal abnormalities and has a non-linear relationship with the blastocyst euploidy rate. Other factors related to both the male and female subjects may only minimally affect this outcome.

Comparison of euploidy rates of blastocysts in women treated with progestins or GnRH antagonist to prevent the luteinizing hormone surge during ovarian stimulation.

STUDY QUESTION: Does the prevalence of euploid blastocysts differ between patients treated with progestin primed ovarian stimulation (PPOS) and those treated with conventional ovarian stimulation? SUMMARY ANSWER: The numbers of blastocysts and euploid blastocysts per patient and the number of euploid embryos per injected oocyte are similar for patients undergoing progestin-primed ovarian stimulation and for those undergoing conventional ovarian stimulation with GnRH antagonist. WHAT IS KNOWN ALREADY: New approaches to ovarian stimulation have been developed based on the use of drugs administrable by mouth instead of via injections. Attention has been dedicated to progestins to block the LH surge. Previous data regarding the number of oocytes retrieved and the number of good-quality embryos generated in PPOS have demonstrated similar outcomes when compared to conventional ovarian stimulation, even if some concerns regarding the quality of embryos have been advanced. STUDY DESIGN, SIZE, DURATION: This is a prospective non-inferiority age-matched case-control study. In a period of 6 months, a total of 785 blastocysts from 1867 injected oocytes obtained from 192 patients were available for analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Infertile women undergoing IVF and preimplanation genetic testing (PGT) cycles were included. Forty-eight patients were treated with PPOS, and for each of them three age-matched historical controls (n = 144) treated with a GnRH antagonist protocol were selected. PGT was performed according to next-generation sequencing technology. MAIN RESULTS AND THE ROLE OF CHANCE: Basal characteristics were similar in the two groups; a substantial similarity of the main outcome measures in the two treatment groups has also been found. The rate of formation of euploid blastocysts per oocyte was 21% in both the two treatment groups. The percentage of patients with euploid embryos and the total number of euploid blastocysts per patient (median and interquartile range, IQR) in the PPOS group were 38.7 (25.5-52.9) and 2 (1.3-3.1), respectively. These figures were not significantly different in women treated with the GnRH antagonist protocol i.e. 42 (28-53.8) and 2.1 (1.3-2.9), respectively. LIMITATIONS, REASONS FOR CAUTION: This was a case-control study which may limit the reliability of the main findings. WIDER IMPLICATIONS OF THE FINDINGS: Our results encourage the use of PPOS, especially for oocyte donation, for fertility preservation and for patients in which total freezing of embryos is foreseen, for those expected to be high responders or candidates for preimplantation genetic testing. However, studies aiming to investigate the effect of PPOS on the live birth rate are warranted. STUDY FUNDING/COMPETING INTEREST(S): None.

Gonadotrophins modulate cell death-related genes expression in human endometrium.

Background Gonadotrophins exert their functions by binding follicle-stimulating hormone receptor (FSHR) or luteinizing hormone and human chorionic gonadotropin receptor (LHCGR) present on endometrium. Within ovaries, FSH induces autophagy and apoptosis of granulosa cells leading to atresia of non-growing follicles, whereas hCG and LH have anti-apoptotic functions. Endometrial cells express functioning gonadotrophin receptors. The objective of this study was to analyze the effect of gonadotrophins on physiology and endometrial cells survival. Materials and methods Collected endometria were incubated for 48 or 72 h with 100 ng/mL of recombinant human FSH (rhFSH), recombinant human LH (rhLH) or highly purified hCG (HPhCG) alone or combined. Controls omitted gonadotrophins. The effect of gonadotrophins on cytochrome P450 family 11 subfamily A polypeptide 1 (CYP11A1), hypoxia inducible factor 1α (HIF1A), and cell-death-related genes expression was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry for microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B) and apoptotic protease activating factor 1 (APAF-1) was performed. Results Gonadotrophins are able to modulate the endometrial cells survival. FSH induced autophagy and apoptosis by increasing the relative expression of MAP1LC3B and FAS receptor. In FSH-treated samples, expression of apoptosis marker APAF-1 was detected and co-localized on autophagic cells. hCG and LH does not modulate the expression of cell-death-related genes while the up-regulation of pro-proliferative epiregulin gene was observed. When combined with FSH, hCG and LH prevent autophagy and apoptosis FSH-induced. Conclusions Different gonadotrophins specifically affect endometrial cells viability differently: FSH promotes autophagy and apoptosis while LH and hCG alone or combined with rhFSH does not.

Gene expression profiles of human granulosa cells treated with bioequivalent doses of corifollitropin alfa (CFA) or recombinant human follicle-stimulating hormone (recFSH).

Using recombinant DNA technologies, a chimeric gene containing the coding sequences of follicle stimulating hormone (FSH) ß-subunit and C-terminal peptide of the human chorionic gonadotrophin (hCG) ß-subunit have been designed to generate a new gonadotrophin named corifollitropin alfa (CFA). CFA has longer elimination half-life and slower rate of absorption compared with FSH, which makes CFA a long-acting hormone employed as a substitute of the recombinant FSH (recFSH) in the controlled ovarian stimulation (COS). The purpose of this study is to compare the gene expression profiles elicited by bioequivalent doses of CFA or recFSH in primary cultures of human granulosa cells (hGCs). Gonadotrophins exert their functions by binding FSH receptors (FSHRs), activating signaling pathways that increase the cyclic adenosine monophosphate (cAMP) intracellular content. Bioequivalence has been defined as the dose/duration of gonadotrophin treatment able to promote the same amount of intracellular cAMP. hGCs were treated with different doses of either gonadotrophin and the cAMP was measured after different incubation times to establish the bioequivalence. Results obtained by comparing the bioequivalent treatments, showed that CFA is more effective than recFSH in inducing aromatase gene expression after 6 and 24 h from the initial stimulation in agreement with its long-acting characteristic.

From a circle to a sphere: the ultrasound imaging of ovarian follicle with 2D and 3D technology.

Ultrasound follicular count (antral follicle count, AFC) is a necessary tool for measuring ovarian reserve, whereby the estimated number of follicles responsive to FSH can predict the number of oocytes retrieved in IVF cycles and may be the basis for individualized ovarian stimulation therapy. Advances in the ultrasound technology have recently lead to the improvement in resolution and quality of the image. Moreover the automatic measurements of follicular diameter by using some specific 3D software seems associated to several advantages when compared to the 2D technique. Examination time is reduced because the ultrasound scan data are stored and can be analyzed in detail at a later time. These data can be reconstructed in any plane, regardless of the original scan plane facilitating the detailed analysis. Another advantage is that this new technique reduces the operator's influence on scan interpretation and objectivity; therefore, interobserver variability is reduced. Using follicular volume obtained with sono AVC as the measure of follicular growth combined with volume-based criteria for the hCG triggering may in the future improve the treatment outcome compared to that achieved with conventional monitoring with follicular diameter. Better knowledge in this area could be helpful to optimize IVF outcome, by refining ovarian stimulation protocols and obtain high quality oocytes.

Evidence for expression and functionality of FSH and LH/hCG receptors in human endometrium.

PURPOSE: Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) mediate intracellular functions by binding their specific protein G-coupled gonadotrophin receptor, respectively FSH receptor (FSHR) and LH/choriogonadotrophin receptor (LHCGR). Whereas the expression of FSHR and LHCGR in mammals was considered gonad-specific and cell-specific, studies identified gonadotrophin receptors in human female extragonadal reproductive tissues. This study aims to demonstrate that gonadotrophin receptors are expressed in endometrium and mediates intracellular functions. METHODS: Collected endometria (n = 12) from healthy patients (mean age of 36 ± 6) were primary cultured for 24 h. The presence of gonadotrophin receptors was evaluated by RT-PCR followed by the sequencing of the resulted amplicons and by immunohistochemistry in original samples. Endometrial primary cultures were treated with increasing concentration (range 0-100 ng/ml) of either recombinant human LH (rhLH) or recombinant human FSH (rhFSH). Endometria controls had gonadotrophin replaced by the same volume of the culture medium. In gonadotrophin-treated samples, it was evaluated the intracellular cyclic adenosine monophosphate (cAMP) content by enzymatic immunoassay and the expression of steroidogenic genes by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). RESULTS: The sequencing of the RT-PCR amplicons confirmed the presence of both gonadotrophin receptors and immunohistochemistry localized them on the membrane of endometrial glands cells throughout the glandular epithelium. The gonadotrophin-receptor complex was able to increase the intracellular cAMP in a dose-response and time-course manner and to induce steroidogenic genes expression. CONCLUSION: This study demonstrates that both gonadotrophin receptors are expressed along the glandular epithelium of endometria and they mediate the effects of gonadotrophins on intracellular functions.

The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro.

PURPOSE: Anti-Müllerian hormone (AMH) and forkhead box L2 (FOXL2) are two pivotal genes expressed in human granulosa cells (hGCs) where both genes share similar inhibitory functions on activation and follicular growth in order to preserve the ovarian follicle reserve. Furthermore, AMH and FOXL2 contribute to inhibit steroidogenesis, decreasing or preventing the activation of gonadotrophin-dependent aromatase CYP19A1 cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The purpose of this study is to evaluate the role of AMH in regulating the expression of FOXL2. METHODS: Primary cultures of hGCs were treated with increasing concentrations of recombinant human AMH (rhAMH; range 10-100 ng/ml) for 3 h. Negative controls were performed using corresponding amounts of AMH vehicle. Total RNA or proteins were purified and quantified by spectrophotometry. FOXL2 and CYP19A1 gene expression, normalized by reference gene ribosomal protein S7 (RpS7), was evaluated by RT-qPCR. Each reaction was repeated in triplicate. Statistical analysis was performed. Extracted proteins were analyzed by immunoblot using anti-FOXL2 and anti-ß-actin as primary antibodies. RESULTS: rhAMH treatments tested did not modulate the basal expression of aromatase CYP19A1 gene. rhAMH (50 ng/ml) was able to increase FOXL2 gene expression and its intracellular content. CONCLUSIONS: This study demonstrated the existence of an AMH-FOXL2 relationship in hGCs. AMH is capable of increasing both gene and protein expression of FOXL2. Because FOXL2 induces AMH transcription, these ovarian factors could be finely regulated by a positive feedback loop mechanism to preserve the ovarian follicle reserve.

Modulation of gonadotrophin induced steroidogenic enzymes in granulosa cells by d-chiroinositol.

BACKGROUND: d-chiroinositol (DCI) is a inositolphosphoglycan (IPG) involved in several cellular functions that control the glucose metabolism. DCI functions as second messenger in the insulin signaling pathway and it is considered an insulin sensitizer since deficiency in tissue availability of DCI were shown to cause insulin resistance (IR). Polycystic ovary syndrome (PCOS) is a pathological condition that is often accompanied with insulin resistance. DCI can positively affects several aspect of PCOS etiology decreasing the total and free testosterone, lowering blood pressure, improving the glucose metabolism and increasing the ovulation frequency. The purpose of this study was to evaluate the effects of DCI and insulin combined with gonadotrophins namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on key steroidogenic enzymes genes regulation, cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and cytochrome P450 side-chain cleavage (P450scc) in primary cultures of human granulosa cells (hGCs). We also investigated whether DCI, being an insulin-sensitizer would be able to counteract the expected stimulator activity of insulin on human granulosa cells (hGCs). METHODS: The study was conducted on primary cultures of hGCs. Gene expression was evaluated by RT-qPCR method. Statistical analysis was performed applying student t-test, as appropriate (P < 0.05) set for statistical significance. RESULTS: DCI is able to reduce the gene expression of CYP19A1, P450scc and insulin-like growth factor 1 receptor (IGF-1R) in dose-response manner. The presence of DCI impaired the increased expression of steroidogenic enzyme genes generated by the insulin treatment in gonadotrophin-stimulated hGCs. CONCLUSIONS: Insulin acts as co-gonadotrophin increasing the expression of steroidogenic enzymes genes in gonadotrophin-stimulated granulosa cells. DCI is an insulin-sensitizer that counteracts this action by reducing the expression of the genes CYP19A1, P450scc and IGF-1R. The ability of DCI to modulate in vitro ovarian activity of insulin could in part explain its beneficial effect when used as treatment for conditions associated to insulin resistance.

A recombinant Sp185/333 protein from the purple sea urchin has multitasking binding activities towards certain microbes and PAMPs.

The purple sea urchin, Strongylocentrotus purpuratus, possesses a sophisticated innate immune system that responds to microbes effectively by swift expression of the highly diverse Sp185/333 gene family. The Sp185/333 proteins are predicted to have anti-pathogen functions based on inducible gene expression and their significant sequence diversity. Sp185/333 proteins are all predicted to be intrinsically disordered and do not exhibit sequence similarities to other known proteins. To test the anti-pathogen hypothesis, a recombinant Sp185/333 protein, rSp0032, was evaluated and found to exhibit specific binding to marine Vibrio diazotrophicus and to Saccharomyces cerevisiae, but not to two Bacillus species. rSp0032 also binds to LPS, ß-1,3-glucan and flagellin but not to peptidoglycan. rSp0032 binding to LPS can be competed by LPS, ß-1,3-glucan and flagellin but not by peptidoglycan. We speculate that the predicted intrinsically disordered structure of rSp0032 may adapt to different conformations in binding to a limited number of PAMPs and pathogens. Given that rSp0032 binds to a range of targets, and that up to 260 different Sp185/333 proteins can be expressed per individual sea urchin, this family of immune response proteins may facilitate effective host protection against a broad array of potential pathogens encountered in the marine environment.

The anti-Müllerian hormone (AMH) acts as a gatekeeper of ovarian steroidogenesis inhibiting the granulosa cell response to both FSH and LH.

PURPOSE: Anti Müllerian Hormone (AMH) has a negative and inhibitory role in many functions of human granulosa-lutein cells (hGCs) including notoriously the reduction of the aromatase CYP19A1 expression induced by follicle-stimulating hormone (FSH). No data have been provided on the possible role of AMH in modulating the response to luteinizing hormone (LH) (alone or combined with FSH) as well as its effect on other enzymes involved in steroidogenesis including aromatase P450scc. The aim of this study was to investigate the role of AMH as regulator of the basal and stimulated steroids production by hGCs. METHODS: Primary culture of hGCs were incubated with hormones AMH, LH, and FSH, alone or in combination. The CYP19A1 and P450scc messenger RNA (mRNA) expression, normalized by housekeeping ribosomal protein S7 (RpS7) gene, was evaluated by reverse transcriptase quantitative PCR (RT-qPCR). Each reaction was repeated in triplicate. Negative controls using corresponding amount of vehicle control for each hormone treatment were performed. RESULT: AMH did not modulate the basal mRNA expression of both aromatase genes at any of the concentrations tested. Meanwhile, the strong mRNA induction of CYP19A1 and P450scc generated by a 24-h gonadotropin treatment (alone and combined) was suppressed by 20 ng/ml AMH added to culture medium. CONCLUSIONS: These findings contribute in clarifying the relationship between hormones regulating the early phase of steroidogenesis confirming that AMH is playing a suppressive role on CYP19A1 expression stimulated by gonadotropin in hGCs. Furthermore, a similar inhibitory effect for AMH was observed on P450scc gene expression when activated by gonadotropin treatment.

Single sea urchin phagocytes express messages of a single sequence from the diverse Sp185/333 gene family in response to bacterial challenge.

Immune systems in animals rely on fast and efficient responses to a wide variety of pathogens. The Sp185/333 gene family in the purple sea urchin, Strongylocentrotus purpuratus, consists of an estimated 50 (±10) members per genome that share a basic gene structure but show high sequence diversity, primarily due to the mosaic appearance of short blocks of sequence called elements. The genes show significantly elevated expression in three subpopulations of phagocytes responding to marine bacteria. The encoded Sp185/333 proteins are highly diverse and have central effector functions in the immune system. In this study we report the Sp185/333 gene expression in single sea urchin phagocytes. Sea urchins challenged with heat-killed marine bacteria resulted in a typical increase in coelomocyte concentration within 24 h, which included an increased proportion of phagocytes expressing Sp185/333 proteins. Phagocyte fractions enriched from coelomocytes were used in limiting dilutions to obtain samples of single cells that were evaluated for Sp185/333 gene expression by nested RT-PCR. Amplicon sequences showed identical or nearly identical Sp185/333 amplicon sequences in single phagocytes with matches to six known Sp185/333 element patterns, including both common and rare element patterns. This suggested that single phagocytes show restricted expression from the Sp185/333 gene family and infers a diverse, flexible, and efficient response to pathogens. This type of expression pattern from a family of immune response genes in single cells has not been identified previously in other invertebrates.

KRP-203, sphingosine 1-phosphate receptor type 1 agonist, ameliorates atherosclerosis in LDL-R-/- mice.

OBJECTIVE: Sphingosine 1-phosphate (S1P) partly accounts for antiatherogenic properties of high-density lipoproteins. We previously demonstrated that FTY720, a synthetic S1P analog targeting all S1P receptors but S1P receptor type 2, inhibits murine atherosclerosis. Here, we addressed the identity of S1P receptor mediating atheroprotective effects of S1P. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice on cholesterol-rich diet were given selective S1P receptor type 1 agonist KRP-203 (3.0 mg/kg per day; 6 and 16 weeks). KRP-203 substantially reduced atherosclerotic lesion formation without affecting plasma lipid concentrations. However, KRP-203 induced lymphopenia, reduced total (CD4(+), CD8(+)) and activated (CD69(+)/CD8(+), CD69(+)/CD4(+)) T cells in peripheral lymphoid organs, and interfered with lymphocyte function, as evidenced by decreased T-cell proliferation and interleukin-2 and interferon-γ production in activated splenocytes. Cyto- and chemokine (tumor necrosis factor-α, regulated and normal T cell expressed and secreted) levels in plasma and aortas were reduced by KRP-203 administration. Moreover, macrophages from KRP-203-treated mice showed reduced expression of activation marker MCH-II and poly(I:C)-elicited production of tumor necrosis factor-α, monocyte chemoattractant protein-1, and interleukin-6. In vitro studies demonstrated that KRP-203 reduced tumor necrosis factor-α, interleukin-6, and interferon-γ-induced protein-10 production; IκB and signal transducer and activator of transcription-1 phosphorylation; and nuclear factor κB and signal transducer and activator of transcription-1 activation in poly(I:C)-, lipopolysaccharide-, or interferon-γ-stimulated bone marrow macrophages, respectively. CONCLUSIONS: Present results demonstrate that activation of S1P signaling pathways inhibit atherosclerosis by modulating lymphocyte and macrophage function and suggest that S1P receptor type 1 at least partially mediates antiatherogenic effects of S1P.

Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells.

The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokines.

Invertebrate immune diversity.

The arms race between hosts and pathogens (and other non-self) drives the molecular diversification of immune response genes in the host. Over long periods of evolutionary time, many different defense strategies have been employed by a wide variety of invertebrates. We review here penaeidins and crustins in crustaceans, the allorecognition system encoded by fuhc, fester and Uncle fester in a colonial tunicate, Dscam and PGRPs in arthropods, FREPs in snails, VCBPs in protochordates, and the Sp185/333 system in the purple sea urchin. Comparisons among immune systems, including those reviewed here have not identified an immune specific regulatory "genetic toolkit", however, repeatedly identified sequences (or "building materials" on which the tools act) are present in a broad range of immune systems. These include a Toll/TLR system, a primitive complement system, an LPS binding protein, and a RAG core/Transib element. Repeatedly identified domains and motifs that function in immune proteins include NACHT, LRR, Ig, death, TIR, lectin domains, and a thioester motif. In addition, there are repeatedly identified mechanisms (or "construction methods") that generate sequence diversity in genes with immune function. These include genomic instability, duplications and/or deletions of sequences and the generation of clusters of similar genes or exons that appear as families, gene recombination, gene conversion, retrotransposition, alternative splicing, multiple alleles for single copy genes, and RNA editing. These commonly employed "materials and methods" for building and maintaining an effective immune system that might have been part of that ancestral system appear now as a fragmented and likely incomplete set, likely due to the rapid evolutionary change (or loss) of host genes that are under pressure to keep pace with pathogen diversity.

Echinoderm immunity.

A survey for immune genes in the genome for the purple sea urchin has shown that the immune system is complex and sophisticated. By inference, immune responses of all echinoderms maybe similar. The immune system is mediated by several types of coelomocytes that are also useful as sensors of environmental stresses. There are a number of large gene families in the purple sea urchin genome that function in immunity and of which at least one appears to employ novel approaches for sequence diversification. Echinoderms have a simpler complement system, a large set of lectin genes and a number of antimicrobial peptides. Profiling the immune genes expressed by coelomocytes and the proteins in the coelomic fluid provide detailed information about immune functions in the sea urchin. The importance of echinoderms in maintaining marine ecosystem stability and the disastrous effects of their removal due to disease will require future collaborations between ecologists and immunologists working towards understanding and preserving marine habitats.

unpaired (upd)-3 expression and other immune-related functions are stimulated by interleukin-8 in Drosophila melanogaster SL2 cell line.

Invertebrate innate immunity relies on both cellular and humoral components. Among humoral factors, there is less information on soluble molecules able to act as signals during the immune response (i.e. cytokines). In Drosophila melanogaster, the cytokine Unpaired (Upd)-3, is known to activate the JAK/STAT pathway, but it is still not clear which molecules and pathways are responsible for its induction and secretion. It has been proposed that highly chemotactic factors may increase the expression of upd-3. In this respect, we have studied the effects of the chemotactic human recombinant (hr) interleukin (IL)-8 on the immune functions of Drosophila SL2 macrophage-like cells. The hrIL-8 increases the percentage of phagocytic cells with a specific timing and enhances the expression of the cytokine, upd-3, as well as that of the putative cytokine Drosophila helical factor (dhf). The antimicrobial peptides defensin, cecropin A1 and diptericin, are all influenced in their expression by the human chemokine, while hrIL-8 leaves unaffected the expression of drosomycin, i.e. the antimicrobial peptide more strictly connected with the Toll pathway. Similar effects to those registered for hrIL-8 are also provoked by a specific activator of the Imd pathway, i.e. the Escherichia coli peptidoglycan. RNAi experiments demonstrated that the silencing of the Imd pathway-associated kinase dTAK1, leaves unaffected the induction of upd-3, while it completely abolishes the effects of hrIL-8-on the expression of dhf. Our data suggest that the Imd pathway is not fundamental in regulating the levels of upd-3, whereas it controls the expression of the putative cytokine dhf.

Stress and immune response in the mussel Mytilus galloprovincialis.

The present study investigates the effects on immune-related parameters of various stress factors (air exposure, mechanical stress, high temperature and extreme salinity conditions) faced by the bivalve mollusc Mytilus galloprovincialis during marketing procedures. We observed that some stress typologies increase phagocytosis and the number of circulating immunocytes, while others can modify immunocyte response towards a further perturbation, i.e. the marine algal toxin yessotoxin. Our results suggest that non-lethal stress can be counteracted for sometime by increasing the level of some defence parameters. Moreover, our data indicate that fishing and transport procedures could interfere with mussel immunosurveillance.