The basis of graft rejection in the earthworms Lumbricus terrestris and Eisenia foetida

Body wall grafts have been exchanged between Lumbricus terrestris and Eisenia foetida unicolor, and the survival times of second-set grafts and a limited number of third-set grafts have been compared with those of first-set grafts. Survival of grafts has also been studied following implantation. The results do not support the hypothesis that second-set rejection is accelerated due to an immune response as in higher vertebrates. Evidence is presented that Eisenia are individually different and that the differential graft survival may be due to tissue incompatibility, survival times depending on the degree of difference in the environment presented by the recipient.     

Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.     

Development of Transplantation Immunity and Restoration Experiments in the Thymectomized Amphibian

This paper examines the thymic dependence of alloimmunity inamphibians. In Xenopus, the presence of a thymus during thefirst 2 weeks of life is essential for the development of normalfirst-set skin allograft immunity. Thymectomy during this earlyperiod always impairs the alloimmune response of young adulttoads. However, most of these thymectomized animals are ableto completely destroy skin allografts, albeit with prolongedrejection times. Chronic graft rejection, rather than tolerance,still occurs following thymectomy as early as 5 days, when thethymus contains no small lymphocytes. In contrast to the considerabledifferences in first-set allograft survival times in controland early-thymectomized Xenopus, second-set grafts, appliedsubsequent to first-set destruction, are rejected in acute fashion(<3 weeks) in both groups. That the defect in first-set alloimmunityis specifically related to absence of thymus has been confirmedby implanting allogeneic thymus 2 weeks post-thymectomy. Thedonor thymus remains healthy and restores the allograft responseto normal. In contrast, allogeneic spleen does not reconstituteand itself often undergoes destruction. Preliminary autoradiographicexperiments on lymphoid tissue involvement in first-set allograftrejection are also described.     

The capacities of earthworms to heal wounds and to destroy allografts are modified by polychlorinated biphenyls (PCB).

Earthworms (Lumbricus terrestris) were maintained at 15 degrees C and exposed on filter paper to 10 micrograms/cm2 of the polychlorinated biphenyl (PCB) Aroclor 1254 for 5 days prior to surgical treatments which consisted of wounds, autografts, and allografts. At 1 day after surgery, we observed a higher percentage of healing defects and a significantly greater number of early signs of allograft rejection in exposed worms. Observations for 25 days post-transplantation revealed no response to autografts, but an acceleration of the allograft rejection process in exposed earthworms. We postulate that Aroclor modified host coelomocytes and/or their interactions associated with antigen recognition and inflammation.     

Coelomocytes of earthworms: the T-cell-like rosette.

The coelomocytes forming spontaneous rosettes with sheep red blood cells (SRBC) were studied under various experimental conditions in lumbricus terrestris. The percentage of rosettes did vary with the pH, increased with the incubation period and remained constant at 4°C and at room temperature. The viability of the coelomocytes was a prerequisite for the formation of rosettes.The phenomenon was found to be stable, repeatable, and specific, thus suggesting rosettes are probably not an artefact.The coelomocytes forming rosettes were basophilic, nongranulated, and nonadhering cells with a large nucleus and little cytoplasm closely resembling vertebrate T-lymphocytes. The adhering cells did not form rosette.These results suggest that the coelomocytes forming rosettes could well be the evolutionary precursors or analogs of immunocyte rosettes forming cells of the vertebrates. Whether or not the coelomocyte receptors for the SRBC are similar to those of the T-cells of the vertebrate remains to be established.     

Survival of body wall autographs, allografts, and xenografts in the earthworm Eisenia foetida.

It is acknowledged that autografts of Eisenia foetida body wall are accepted by their hosts. Allografts, however, have not been found to be accepted, although macroscopic observation of these might suggest they are, possibly owing to fusion of grafts and host epidermal cells. All xenografts eventually fail. Histological studies show that both allografts and xenografts were isolated from hosts, invaded, absorbed, and replaced with collagen. Statistical analyses of results from multiple transfers of xenografts show no evidence of hosts producing a heightened response of any nature to a second challenge of the same antigen.     

Hemolymph functions in Mytilus californianus: The cytochemistry of hemocytes and their responses to foreign implants and hemolymph factors in phagocytosis

The hemocytes of Mytilus californianus are of three types: small and large basophils and large granular acidophils. The basophils contain lysosomal enzymes and phagocytose colloidal carbon. Agglutinins for yeast and human A Rh+ve erythrocytes are present in plasma, but are not needed for effective phagocytosis; in vitro both acidophilic and basophilic hemocytes rapidly phagocytose these particles. Plasma proteins, analyzed electrophoretically, are under strong homeostatic control. When Mya arenaria mantle is placed orthotopically on M. californianus mantle, the implant is invaded by host hemocytes in a manner consistent with that described in other published reports on molluscan graft rejection. Steady state is achieved by 26 days postimplant. Second- and third-set implants are rejected more rapidly than are first-set implants, but this is not a specific response. Third-set implants elicit a host cellular response that is more localized than the response to first-set implants. These data do not permit conclusions with respect to memory in these molluscan immune responses, but do imply a qualitative “improvement” in this quasi-immune response of M. californianus.     

Ultrastructure of coelomic epithelium and coelomocytes of the starfish Asterias rubens L. in norm and after wounding

Samples of coelomic epithelium (CE) and coelomocyte suspension of intact and wounded starfish Asterias rubens L. were studied by electron microscopy. The CE was shown to be composed of three types of cells: flagellar (approximately 60%), secretory (approximately 3%), and myoepithelial (approximately 37%); flagellar and secretory cells form the CE apical surface. Secretory cells are represented by two subtypes, i.e., granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. In 4–5% of cases, adjacent flagellar cells are separated by various sizes of intercellular gaps. These gaps seem to be lacunae left by the flagellar cells after their release into the coelomic cavity. The morphological pattern of the conversion of CE flagellar cells into coelomocytes was characterized. After a moderate wounding used in the present study, no significant structural alterations in the CE organization were revealed. In coelomocyte suspension, small rounded young coelomocytes (approximately 3%) and the larger mature coelomocytes (approximately 97%) were found. On the surface of one of the young coelomocytes, a flagellum was revealed. Surface of the mature coelomocytes forms processes of various size and structure; their cytoplasm contains lysosomes and phagocytic vacuoles of different size. After wounding, a coelomocyte activation was found that consisted of a sharp rise in the number and length of filopodia on their surface, as well as the formation of multicellular aggregates. The complex of ultrastructural data allows it to be suggested that the histogenesis of coelomocytes from CE flagellar cells is a process of cell transdifferentiation.     

Calcineurin regulates coelomocyte endocytosis via DYN-1 and CUP-4 in Caenorhabditis elegans

C. elegans coelomocytes are macrophage-like scavenger cells that provide an excellent in vivo system for the study of clathrin-mediated endocytosis. Using this in vivo system, several genes involved in coelomocyte endocytosis have been identified previously. However, the detailed mechanism of endocytic pathway is still unknown. Here, we report a new function of calcineurin, an evolutionarily conserved Ca²⁺/calmodulin-dependent Ser/Thr protein phosphatase, in coelomocyte endocytosis. We found that calcineurin mutants show defective coelomocyte endocytosis. Genetic analysis suggests that calcineurin and a GTPase, dynamin (DYN-1), may function upstream of an orphan receptor, CUP-4, to regulate endocytosis. Therefore, we propose a model in which calcineurin may regulate coelomocyte endocytosis via DYN-1 and CUP-4 in C. elegans.     

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin,Paracentrotus lividus,using capillary LC-ESI-MS/MS

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac₂, Neu5,7Ac₂, Neu5,8Ac₂, Neu5,7,9Ac₃, Neu5Gc7,9Ac₂, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).     

A Randomized and Controlled Multicenter Prospective Study of the Chinese Medicinal Compound Fufang Xuelian Burn Ointment for the Treatment of Superficial and Deep Second-Degree Burn Wounds

The objective of this study was to evaluate the efficacy and safety of a traditional Chinese medicine, Fufang Xuelian Burn Ointment (FXBO), to treat superficial and deep second-degree burn wounds. A four-center, randomized, controlled, and prospective study was conducted. Overall, 240 patients with either superficial or deep second-degree burn wounds were enrolled consecutively in this study. Patients who were randomly assigned to the control group (superficial: 72, deep: 48) underwent common burn wound therapy, whereas those randomized to the treatment group (superficial: 72, deep: 48) received common burn wound therapy plus topical FXBO. The healing rate, healing time, effective rate, and safety data were compared between the two groups. The baseline characteristics were comparable for the two groups. The healing rate was 94.79(±7.50) in the control group and 98.60(±5.69) in the FXBO group after 14 days for patients with superficial second-degree burn wounds (P = 0.000), and 95.17(±9.68) versus 97.44(±9.81) at 28 for deep second-degree burn wounds (P = 0.025). The median healing time in the FXBO group were 9 and 21 days for superficial and deep second-degree burns, respectively, compared to 10.5 and 22.5 days, respectively, in control group (P _superficial = 0.000 and P _deep = 0.009). The results of the effective rate showed that comprehensive efficacy of the FXBO group was improved compared to the control group for either superficial or deep second-degree burns (P _superficial = 0.035 and P _deep = 0.003). There were no reported drug-related adverse events in both groups. Therefore, FXBO was well tolerated and more effective than control group for treating superficial and deep second-degree burn wounds.     

Magnetic Resonance Imaging (MRI) of Intratumoral Voxel Heterogeneity as a Potential Response Biomarker: Assessment in a HER2+ Esophageal Adenocarcinoma Xenograft Following Trastuzumab and/or Cisplatin Therapy

We evaluated magnetic resonance imaging (MRI) voxel heterogeneity following trastuzumab and/or cisplatin in a HER2+ esophageal xenograft (OE19) as a potential response biomarker. OE19 xenografts treated with saline (controls), monotherapy, or combined cisplatin and trastuzumab underwent 9.4-T MRI. Tumor MRI parametric maps of T1 relaxation time (pre/post contrast), T2 relaxation time, T2* relaxation rate (R2*), and apparent diffusion coefficient obtained before (TIME0), after 24 hours (TIME1), and after 2 weeks of treatment (TIME2) were analyzed. Voxel histogram and fractal parameters (from the whole tumor, rim and center, and as a ratio of rim‐to‐center) were derived. Tumors were stained for immunohistochemical markers of hypoxia (CA-IX), angiogenesis (CD34), and proliferation (Ki-67). Combination therapy reduced xenograft growth rate (relative change, ? +0.58 ± 0.43 versus controls, ? +4.1 ± 1.0; P = 0.008). More spatially homogeneous voxel distribution between the rim to center was noted after treatment for combination therapy versus controls, respectively, for contrast-enhanced T1 relaxation time (90th percentile: ratio 1.00 versus 0.88, P = 0.009), T2 relaxation time (mean: 1.00 versus 0.92, P = 0.006; median: 0.98 versus 0.91, P = 0.006; 75th percentile: 1.02 versus 0.94, P = 0.007), and R2* (10th percentile: 0.99 versus 1.26, P = 0.003). We found that combination and trastuzumab monotherapy reduced MRI spatial heterogeneity and growth rate compared to the control or cisplatin groups, the former providing adjunctive tumor response information.     

Phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin Lytechinus variegatus (Lamarck, 1816)

The echinoderms are deuterostomic animals with a nonspecific immune system similar to that of vertebrates. Among coelomocytes, phagocytic amoebocytes have a key role in the nonspecific immune response in sea urchin, being responsible for microorganisms elimination through phagocytosis and also for humoral secretions of a wide spectrum. Sub-populations of phagocytic amoebocytes (PA) have been previously described and two distinct sub populations in the oral (OR) and aboral (AB) regions of the perivisceral coelom of L.variegatus in the present study were found. In the OR there is a higher number of PA with higher phagocytic capacity after 30 minutes of incubation with yeast and higher percentage of intranuclear iron crystalloids. The germicide capacity under the fluorescence technique did not show any difference. SDS-PAGE analysis showed different protein patterns between coelomocytes of OR and AB. Gravitational force had no effect in PA distribution and no physical barrier was found in the perivisceral coelom. The other coelomocyte (vibratile cells, red spherulocytes and white spherulocytes) populations were not different in OR compared with AB in their distribution. Some aspects of the possible causes of the differences found for PA are discussed in the paper.     

Ultrastructure of the Coelomocytes in the Tentacular Coelom of Thysanocardia nigra Ikeda, 1904 (Sipuncula)

Free-floating coelomocytes in the tentacular coelomic cavity of the sipunculan Thysanocardia nigra Ikeda, 1904, were studied using light interference contrast microscopy and scanning and transmission electron microscopy. The following coelomocyte types were distinguished: hemerythrocytes, amoebocytes, and two morphological types of granular cells. No clusters of specialized cells that had been reported to occur in the trunk coelom of Th. nigra were found in the tentacular coelom. The corresponding types of coelomocytes from the tentacular and trunk coelomic cavities were shown to differ in size. These two coeloms are completely separated in sipunculans.     

Alloimmune memory is absent in the Red Sea hydrocoral Millepora dichotoma

When two allogeneic colonies of the Red Sea hydrocoral Millepora dichotoma (Cnidaria, Hydrozoa) come into tissue contact, one of the genotypes is usually overgrown by the other. The directionality and pace of this alloresponse are thought to be genetically determined. We established tissue contacts between allogeneic colonies in situ in order to elucidate a possible memory component in this response. First-set interactions were established from all possible pairwise combinations between three colonies in eight replicates per combination. Interactions were followed up for 8 weeks. Thereafter, interacting pairs were detached and either regrafted near the original contact area to form second-set assays or challenged by third party grafts. Additional delayed first-set assays was also established. Overgrowth of delayed first-set, second-set, and third-party grafts was followed again for 8 weeks. The mean overgrowths recorded in the second set of the interactions were indistinguishable from the first sets in all three colony combinations. A specific alloimmune memory has not been found in this cnidarian system as opposed to other cases within the phylum.     

Intermediate filament proteins in echinoderm coelomocytes

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droebachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single M_r 68 000 sea urchin lamin, as well as two putative lamin isoforms of approximately M_r 70 000 and 68 000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of M_r 46 000 and 58 000 polypeptides, while M_r 215 000 and 185 000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.     

Cellular biomarkers to elucidate global warming effects on Antarctic sea urchin Sterechinus neumayeri

Global warming is a reality and its effects have been widely studied. However, the consequences for marine invertebrates remain poorly understood. Thus, the present study proposed to evaluate the effect of elevated temperature on the innate immune system of Antarctic sea urchin Sterechinus neumayeri. Sea urchins were collected nearby Brazilian Antarctic Station ??Comandante Ferraz?? and exposed to 0 (control), 2 and 4°C for periods of 48?h, 2, 7 and 14?days. After the experimental periods, coelomic fluid was collected in order to perform the following analyses: coelomocytes differential counting, phagocytic response, adhesion and spreading coelomocytes assay, intranuclear iron crystalloid and ultra structural analysis of coelomocytes. The red sphere cell was considered a biomarker for heat stress, as they increased in acute stress. Besides that, a significant increase in phagocytic indexes was observed at 2°C coinciding with a significant increase of intranuclear iron crystalloid at the same temperature and same time period. Furthermore, significant alterations in cell adhesion and spreading were observed in elevated temperatures. The ultra structural analysis of coelomocytes showed no significant difference across treatments. This was the first time that innate immune response alterations were observed in response to elevated temperature in a Polar echinoid.     

Xenograft Tumors Vascularized with Murine Blood Vessels May Overestimate the Effect of Anti-Tumor Drugs: A Pilot Study

Recent evidence demonstrated that endothelial cells initiate signaling events that enhance tumor cell survival, proliferation, invasion, and tumor recurrence. Under this new paradigm for cellular crosstalk within the tumor microenvironment, the origin of endothelial cells and tumor cells may have a direct impact on the pathobiology of cancer. The purpose of this pilot study was to evaluate the effect of endothelial cell species (i.e. murine or human) on xenograft tumor growth and response to therapy. Tumor xenografts vascularized either with human or with murine microvascular endothelial cells were engineered, side-by-side, subcutaneously in the dorsum of immunodefficient mice. When tumors reached 200 mm³, mice were treated for 30 days with either 4 mg/kg cisplatin (i.p.) every 5 days or with 40 mg/kg sunitinib (p.o.) daily. Xenograft human tumors vascularized with human endothelial cells grow faster than xenograft tumors vascularized with mouse endothelial cells (P<0.05). Notably, human tumors vascularized with human endothelial cells exhibited nuclear translocation of p65 (indicative of high NF-kB activity), and were more resistant to treatment with cisplatin or sunitinib than the contralateral tumors vascularized with murine endothelial cells (P<0.05). Collectively, these studies suggest that the species of endothelial cells has a direct impact on xenograft tumor growth and response to treatment with the chemotherapeutic drug cisplatin or with the anti-angiogenic drug sunitinib.     

Fertility of sows injected with exogenous oestradiol and/or gonadotrophins to control post-weaning oestrus

In the first of two experiments 28 multiparous sows were allocated to one of the following treatments 2 days after weaning at approximately 35 days post partum: (1) untreated; (2) i.m. injection 10 μg oestradiol benzoate (OB)/kg body weight (b.wt.); and (3) i.m. injection 20 μg OB/kg b.wt. Sows were bred at first post-weaning oestrus and ovulation rate assessed at slaughter. The mean interval from weaning to oestrus in each group was: (1) 5.6 ± 0.2; (2) 4.7 ± 0.2; and (3) 4.7 ± 0.2 days; the mean ovulation rates in groups 1 and 2 (18.7 ± 0.6 and 17.4 ± 1.8, respectively) were significantly higher (P < 0.01) than that of 12.0 ± 1.7 for treatment 3 sows. Two untreated and one each of the treated sows were not cycling at slaughter.In the second experiment 75 multiparous sows weaned at 28 ± 3 days post partum (day 0) were evenly allocated with respect to parity to one of four treatment groups: (1) untreated; (2) i.m. injection 10 μg OB/kg b.wt. on day 2; (3) PG600 (400 iu PMSG + 200 iu hCG) injection subcutaneous day 0; and (4) combined PG600/OB treatment as in (2) and (3) above. Sows were bred naturally at the first post-weaning oestrus and fertility assessed at farrowing. Control animals had a significantly longer (P < 0.05) weaning to oestrus interval (4.53 ± 0.25 days) compared to treatment 2 (4.03 ± 0.13) treatment 3 (3.97 ± 0.12) and treatment 4 (3.81 ± 0.07) sows. Sows treated with PG600 alone showed a significant increase (P < 0.05) in numbers born live compared to pre-treatment values. A smaller and non-significant increase in numbers born live in control sows (probably related to increasing parity) was not observed in either OB- or PG600/OB-treated animals.These results suggest that with further modification of the treatments, a system may be developed for introducing fixed-time artificial insemination (AI) or mating as a means of controlling the reproductive performance of the weaned sow.