Protect Host Cells Research Articles

Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast

The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4–5nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments.

Inhibition of viral protein translation by indomethacin in vesicular stomatitis virus infection: role of eIF2α kinase PKR.

SummaryIndomethacin, a cyclooxygenase‐1 and ‐2 inhibitor widely used in the clinic for its potent anti‐inflammatory/analgesic properties, possesses antiviral activity against several viral pathogens; however, the mechanism of antiviral action remains elusive. We have recently shown that indomethacin activates the double‐stranded RNA (dsRNA)‐dependent protein kinase R (PKR) in human colon cancer cells. Because of the important role of PKR in the cellular defence response against viral infection, herein we investigated the effect of indomethacin on PKR activity during infection with the prototype rhabdovirus vesicular stomatitis virus. Indomethacin was found to activate PKR in an interferon‐ and dsRNA‐independent manner, causing rapid (< 5 min) phosphorylation of eukaryotic initiation factor‐2 α‐subunit (eIF2α). These events resulted in shutting off viral protein translation and blocking viral replication (IC 50 = 2 μM) while protecting host cells from virus‐induced damage. Indomethacin did not affect eIF2α kinases PKR‐like endoplasmic reticulum‐resident protein kinase (PERK) and general control non‐derepressible‐2 (GCN2) kinase, and was unable to trigger eIF2α phosphorylation in the presence of PKR inhibitor 2‐aminopurine. In addition, small‐interfering RNA‐mediated PKR gene silencing dampened the antiviral effect in indomethacin‐treated cells. The results identify PKR as a critical target for the antiviral activity of indomethacin and indicate that eIF2α phosphorylation could be a key element in the broad spectrum antiviral activity of the drug.

Two Duplicated Genes DDI2 and DDI3 in Budding Yeast Encode a Cyanamide Hydratase and Are Induced by Cyanamide

Two DNA damage-inducible genes in Saccharomyces cerevisiae, DDI2 and DDI3, are identical and encode putative HD domain-containing proteins, whose functions are currently unknown. Because Ddi2/3 also shows limited homology to a fungal cyanamide hydratase that converts cyanamide to urea, we tested the enzymatic activity of recombinant Ddi2. To this end, we developed a novel enzymatic assay and determined that the Km value of the recombinant Ddi2/3 for cyanamide is 17.3 ± 0.05 mm, and its activity requires conserved residues in the HD domain. Unlike most other DNA damage-inducible genes, DDI2/3 is only induced by a specific set of alkylating agents and surprisingly is strongly induced by cyanamide. To characterize the biological function of DDI2/3, we sequentially deleted both DDI genes and found that the double mutant was unable to metabolize cyanamide and became much more sensitive to growth inhibition by cyanamide, suggesting that the DDI2/3 genes protect host cells from cyanamide toxicity. Despite the physiological relevance of the cyanamide induction, DDI2/3 is not involved in its own transcriptional regulation. The significance of cyanamide hydratase activity and its induced expression is discussed.

The role of unanchored polyubiquitin chains and the TRIM E3-ubiquitin ligase family of proteins in the innate immune response to influenza virus infection (INM3P.402)

Abstract Intracellular innate immune responses are essential to protect host cells against pathogens. Upon infection, pattern recognition receptors detect incoming microbes and trigger downstream signaling pathways leading to production of antiviral type-I interferons (IFNs).These signaling pathways are regulated by different posttranslational modifications including the ubiquitin (Ub) system. Proteins covalently attached to K48-linked polyUb are targeted for degradation by the proteasome, whereas protein modification with K63-linked polyUb may have non-proteolytic activating functions. Unanchored Ub chains that are not covalently attached to any protein are also important for kinase activation. Tripartite motif (TRIM) proteins, which function as E3-ubiquitin ligases, have been implicated in antiviral activity. We found that TRIM6 is important in the synthesis of unanchored K48-linked polyUb chains, which activate the IKKε kinase for STAT1 phosphorylation and induction of an antiviral response. These polyUb chains and IKKε interact in vivo in an IFN-I signaling dependent manner upon virus infection in mice. To better understand the physiological role of unanchored polyUb during virus infection, we developed a system to isolate ubiquitin-interacting proteins from the lungs of influenza-infected mice. Using a proteomics approach we identified new cellular factors that interact with unanchored polyUb chains in vivo and may be involved in the innate immune antiviral response.

Inflammation: Live long and prosper with Siglecs.

Siglec receptors may support longevity in mammals by protecting host cells against damaging free radicals.

Mechanism for glutathione-mediated protection against the Pseudomonas aeruginosa redox toxin, pyocyanin

Pseudomonas aeruginosa is an important human pathogen associated with several acute and chronic conditions, including diseases of the airways and wounds. The organism produces pyocyanin, an extracellular redox toxin that induces oxidative stress, depletes intracellular glutathione (GSH) and induces proliferative arrest and apoptosis, thus compromising the ability of tissue to repair itself. GSH is an important intra- and extracellular antioxidant, redox buffer and detoxifies xenobiotics by increasing their polarity, which facilitates their elimination. As previous studies have reported exogenous GSH to be protective against pyocyanin toxicity, this study was undertaken to explore the mechanism by which GSH protects host cells from the deleterious effects of the toxin. Co-incubation of pyocyanin with GSH resulted in a time-dependent diminished recovery of the toxin from the incubation medium. Concurrently, a highly polar green-colored metabolite was recovered that exhibited a UV–visible spectrum similar to pyocyanin and which was determined by mass spectrometry to have a major ion (m/z=516) consistent with a glutathione conjugate. The ability of the conjugate to oxidize NADPH and to reduce molecular oxygen with the production of reactive oxygen species was comparable to pyocyanin yet it no longer demonstrated cytotoxicity towards host cells. These data suggest that GSH forms a cell-impermeant conjugate with pyocyanin and that availability of the thiol may be critical to minimizing the toxicity of this important bacterial virulence factor at infection sites. Our data indicate that for GSH to have a clinically effective role in neutralizing pyocyanin, the thiol needs to be available at millimolar concentrations.

A CD46-like molecule functional in teleost fish represents an ancestral form of membrane-bound regulators of complement activation.

In the complement system, the regulators of complement activation (RCA) play crucial roles in controlling excessive complement activation and in protecting host cell from misdirected attack of complement. Several members of RCA family have been cloned from cyclostome and bony fish species and classified into soluble and membrane-bound type as in mammalian RCA factors. Complement-regulatory functions have been described only for soluble RCA of lamprey and barred sand bass; however, little is known on the biological function of the membrane-bound RCA proteins in the lower vertebrates. In this study, a membrane-bound RCA protein, designated teleost complement-regulatory membrane protein (Tecrem), was cloned and characterized for its complement-regulatory roles. Carp Tecrem, an ortholog of a zebrafish type 2 RCA, ZCR1, consists of four short consensus repeat modules, a serine/threonine/proline-rich domain, a transmembrane region, and a cytoplasmic domain, from the N terminus, as does mammalian CD46. Tecrem showed a ubiquitous mRNA expression in carp tissues, agreeing well with the putative regulatory role in complement activation. A recombinant Chinese hamster ovary cell line bearing carp Tecrem showed a significantly higher tolerance against lytic activity of carp complement and less deposition of C3-S, the major C3 isotypes acting on the target cell, than control Chinese hamster ovary (mock transfectant). Anti-Tecrem mAb enhanced the depositions of carp C3 and two C4 isotypes on autologous erythrocytes. Thus, the present findings provide the evidence of complement regulation by a membrane-bound group 2 RCA in bony fish, implying the host-cell protection is an evolutionarily conserved mechanism in regulation of the complement system.

NS5B induces up-regulation of the BH3-only protein, BIK, essential for the hepatitis C virus RNA replication and viral release

Hepatitis C virus (HCV) induces cytopathic effects in the form of hepatocytes apoptosis thought to be resulted from the interaction between viral proteins and host factors. Using pathway specific PCR array, we identified 9 apoptosis-related genes that are dysregulated during HCV infection, of which the BH3-only pro-apoptotic Bcl-2 family protein, BIK, was consistently up-regulated at the mRNA and protein levels. Depletion of BIK protected host cells from HCV-induced caspase-3/7 activation but not the inhibitory effect of HCV on cell viability. Furthermore, viral RNA replication and release were significantly suppressed in BIK-depleted cells and over-expression of the RNA-dependent RNA polymerase, NS5B, was able to induce BIK expression. Immunofluorescence and co-immunoprecipitation assays showed co-localization and interaction of BIK and NS5B, suggesting that BIK may be interacting with the HCV replication complex through NS5B. These results imply that BIK is essential for HCV replication and that NS5B is able to induce BIK expression.

Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion.

Mycobacterium tuberculosis (Mtb) requires protein secretion systems like ESX-1 for intracellular survival and virulence. The major virulence determinant and ESX-1 substrate, EsxA, arrests phagosome maturation and lyses cell membranes, resulting in tissue damage and necrosis that promotes pathogen spread. To identify inhibitors of Mtb protein secretion, we developed a fibroblast survival assay exploiting this phenotype and selected molecules that protect host cells from Mtb-induced lysis without being bactericidal in vitro. Hit compounds blocked EsxA secretion and promoted phagosome maturation in macrophages, thus reducing bacterial loads. Target identification studies led to the discovery of BTP15, a benzothiophene inhibitor of the histidine kinase MprB that indirectly regulates ESX-1, and BBH7, a benzyloxybenzylidene-hydrazine compound. BBH7 affects Mtb metal-ion homeostasis and revealed zinc stress as an activating signal for EsxA secretion. This screening approach extends the target spectrum of small molecule libraries and will help tackle the mounting problem of antibiotic-resistant mycobacteria.

Identification and characterization of two novel spliced genes located in the orf47-orf46-orf45 gene locus of Kaposi's sarcoma-associated herpesvirus.

The orf47-orf46-orf45 gene cluster of Kaposi's sarcoma-associated herpesvirus (KSHV) is known to serially encode glycoprotein L (gL), uracil DNA glycosylase, and a viral tegument protein. Here, we identify two novel mRNA variants, orf47/45-A and orf47/45-B, alternatively spliced from a tricistronic orf47-orf46-orf45 mRNA that is expressed in the orf47-orf46-orf45 gene locus during the early stages of viral reactivation. The spliced gene products, ORF47/45-A and ORF47/45-B, consist of only a partial region of gL (ORF47), a unique 7-amino-acid motif, and the complete tegument protein ORF45. Like the ORF45 protein, ORF47/45-A and ORF47/45-B expressed in cells sufficiently activate the phosphorylation of p90 ribosomal S6 kinase (RSK) and extracellular signal-regulated protein kinase (ERK). However, unlike ORF45, both ORF47/45-A and ORF47/45-B contain a signal peptide sequence and are localized at the endoplasmic reticulum (ER). Additionally, we found that ORF47/45-A and ORF47/45-B have an extra function that mediates the upregulation of GRP78, a master regulator of ER homeostasis. The important event regarding GRP78 upregulation can be observed in all tested KSHV-positive cell lines after viral reactivation, and knockdown of GRP78 in cells significantly impairs viral lytic cycle progression, especially at late lytic stages. Compared with some other viral glycoproteins synthesized through the ER, our results strongly implicate that the ORF47/45 proteins may serve as key effectors for controlling GRP78 expression and ER homeostasis in cells. Taken together, our findings provide evidence showing the reciprocal association between the modulation of ER homeostasis and the progression of the KSHV lytic cycle. Emerging evidence has shown that several viruses appear to use different strategies to control ER homeostasis for supporting their productive infections. The two parts of this study identify two aspects of the association between the regulation of ER homeostasis and the progression of the KSHV lytic cycle. The first part characterizes the function of two early lytic cycle proteins, ORF47/45-A and ORF47/45-B, on the activation of a major ER chaperone protein, GRP78. In addition to the ability to promote GRP78 upregulation, the ORF47/45 proteins also activate the phosphorylation of RSK and ERK. The second part reveals that upregulation of GRP78 is essential for the progression of the KSHV lytic cycle, especially at late stages. We therefore propose that activation of GRP78 expression by viral proteins at the early lytic stage may aid with the protection of host cells from severe ER stress and may directly involve the assembly or release of virions.

Protective effect of fluvastatin on influenza virus infection

Statins are 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors and have pleiotropic effects. It has been suggested that statins may be a potential treatment during the next influenza pandemic. In a previous study we found that a statin/caffeine combination protects BALB/c mice against Influenza A, subtypes haemagglutinin type 5 and neuraminidase type 1 (H5N1), H3N2 and H1N1 infection. The effect of statins alone on influenza virus infection, however, is not known. In this study, it was investigated whether fluvastatin is capable of inhibiting influenza A virus replication in vitro. The results demonstrated that the synthesis of viral RNA and protein was affected by fluvastatin treatment. Virus production was markedly reduced when fluvastatin was administered simultaneously with the virus; however, a greater inhibition was observed when fluvastatin was added following viral adsorption. The selectivity index [SI; 50% cytotoxic concentration (CC50)/50% inhibition concentration (IC50)], however, was only 21. It was further demonstrated that fluvastatin protects host cells against influenza-induced inflammation by reducing the production of tumour necrosis factor-α, interleukin 8 and interferon γ. In conclusion, the results demonstrated that fluvastatin exerted a minor inhibitory effect on influenza virus infection, which involved anti-inflammatory activities.

The Wnt/β-catenin signaling pathway controls the inflammatory response in infections caused by pathogenic bacteria.

Innate immunity against pathogenic bacteria is critical to protect host cells from invasion and infection as well as to develop an appropriate adaptive immune response. During bacterial infection, different signaling transduction pathways control the expression of a wide range of genes that orchestrate a number of molecular and cellular events to eliminate the invading microorganisms and regulate inflammation. The inflammatory response must be tightly regulated because uncontrolled inflammation may lead to tissue injury. Among the many signaling pathways activated, the canonical Wnt/β-catenin has been recently shown to play an important role in the expression of several inflammatory molecules during bacterial infections. Our main goal in this review is to discuss the mechanism used by several pathogenic bacteria to modulate the inflammatory response through the Wnt/β-catenin signaling pathway. We think that a deep insight into the role of Wnt/β-catenin signaling in the inflammation may open new venues for biotechnological approaches designed to control bacterial infectious diseases.

P33 (gC1q Receptor) Prevents Cell Damage by Blocking the Cytolytic Activity of Antimicrobial Peptides

The innate immune system is the first line of defense against invading microbes. Its specificity relies a great deal on host pattern recognition molecules that sense pathogen-associated molecular patterns of the invading pathogen. However, full protection is not always guaranteed, and some early defense mechanisms involved in bacterial killing, such as the complement system, can also exert cytolytic activity against host cells. Although these cascades are tightly regulated, the host has to take additional precautions to prevent its cell destruction. In this study, we describe that p33, a negatively charged surface protein found on endothelial cells also known as gC1q receptor, protects host cells from a cytolytic attack by antimicrobial peptides (AMPs), such as LL37 and β-defensin 3. To this end, we characterized the interaction of p33 with AMPs by biochemical and functional means. Our data show that p33 forms a doughnut-shaped trimer that can bind up to three AMPs, and we identified a segment in p33 forming a β-sheet that mediates the binding to all AMPs. Moreover, our results show that p33 abolishes the lytic activity of AMPs at an equimolar ratio, and it protects endothelial cells and erythrocytes from AMP-induced lysis. Taken together, our data suggest a novel protective mechanism of p33 in modulating innate immune response by neutralizing cytotoxic AMPs at the host cell surface.

The p38 MAPK and JNK Pathways Protect Host Cells against Clostridium perfringens Beta-Toxin

Clostridium perfringens beta-toxin is an important agent of necrotic enteritis and enterotoxemia. Beta-toxin is a pore-forming toxin (PFT) that causes cytotoxicity. Two mitogen-activated protein kinase (MAPK) pathways (p38 and c-Jun N-terminal kinase [JNK]-like) provide cellular defense against various stresses. To investigate the role of the MAPK pathways in the toxic effect of beta-toxin, we examined cytotoxicity in five cell lines. Beta-toxin induced cytotoxicity in cells in the following order: THP-1 = U937 > HL-60 > BALL-1 = MOLT-4. In THP-1 cells, beta-toxin formed oligomers on lipid rafts in membranes and induced the efflux of K(+) from THP-1 cells in a dose- and time-dependent manner. The phosphorylation of p38 MAPK and JNK occurred in response to an attack by beta-toxin. p38 MAPK (SB203580) and JNK (SP600125) inhibitors enhanced toxin-induced cell death. Incubation in K(+)-free medium intensified p38 MAPK activation and cell death induced by the toxin, while incubation in K(+)-high medium prevented those effects. While streptolysin O (SLO) reportedly activates p38 MAPK via reactive oxygen species (ROS), we showed that this pathway did not play a major role in p38 phosphorylation in beta-toxin-treated cells. Therefore, we propose that beta-toxin induces activation of the MAPK pathway to promote host cell survival.

Upregulation of Nrf2 expression by human cytomegalovirus infection protects host cells from oxidative stress

NF-E2 related factor 2 (Nrf2) is a transcription factor that plays a key role(s) in cellular defence against oxidative stress. In this study, we showed that the expression of Nrf2 was upregulated in primary human foreskin fibroblasts (HFFs), following human cytomegalovirus (HCMV/HHV-5) infection. The expression of haem oxygenase-1, a downstream target of Nrf2, was also increased by HCMV infection, and this induction was suppressed in HFFs expressing a small hairpin RNA (shRNA) against Nrf2. The HCMV-mediated increase in Nrf2 expression was abolished when UV-irradiated virus was used or when the activity of casein kinase 2 was inhibited. Host cells infected by HCMV had higher survival rates following oxidative stress induced by buthionine sulfoximine compared with uninfected control cells, but this cell-protective effect was abolished by the use of Nrf2 shRNA. Our results suggest that HCMV-mediated activation of Nrf2 might be beneficial to the virus by increasing the host cell's ability to cope with oxidative stress resulting from viral infection and/or inflammation.

Neutrophils Recruited to Sites of Infection Protect from Virus Challenge by Releasing Neutrophil Extracellular Traps

Neutrophils mediate bacterial clearance through various mechanisms, including the release of mesh-like DNA structures or neutrophil extracellular traps (NETs) that capture bacteria. Although neutrophils are also recruited to sites of viral infection, their role in antiviral innate immunity is less clear. We show that systemic administration of virus analogs or poxvirus infection induces neutrophil recruitment to the liver microvasculature and the release of NETs that protect host cells from virus infection. After systemic intravenous poxvirus challenge, mice exhibit thrombocytopenia and the recruitment of both neutrophils and platelets to the liver vasculature. Circulating platelets interact with, roll along, and adhere to the surface of adherent neutrophils, forming large, dynamic aggregates. These interactions facilitate the release of NETs within the liver vasculature that are able to protect host cells from poxvirus infection. These findings highlight the role of NETs and early tissue-wide responses in preventing viral infection.

Malaria Parasites Co-opt Human Factor H to Prevent Complement-Mediated Lysis in the Mosquito Midgut

Human complement is a first line defense against infection in which circulating proteins initiate an enzyme cascade on the microbial surface that leads to phagocytosis and lysis. Various pathogens evade complement recognition by binding to regulator proteins that protect host cells from complement activation. We show that emerging gametes of the malaria parasite Plasmodium falciparum bind the host complement regulator factor H (FH) following transmission to the mosquito to protect from complement-mediated lysis by the blood meal. Human complement is active in the mosquito midgut for approximately 1 hr postfeeding. During this period, the gamete surface protein PfGAP50 binds to FH and uses surface-bound FH to inactivate the complement protein C3b. Loss of FH-mediated protection, either through neutralization of FH or blockade of PfGAP50, significantly impairs gametogenesis and inhibits parasite transmission to the mosquito. Thus, Plasmodium co-opts the protective host protein FH to evade complement-mediated lysis within the mosquito midgut.

Absence of CD59 Exacerbates Systemic Autoimmunity in MRL/lpr Mice

CD59 is a GPI-anchored membrane regulator of complement expressed on blood cells as well as peripheral tissues. It protects host cells from complement injury by inhibiting formation of the membrane attack complex. Recent studies in mice have suggested also a role of CD59 in T cell immune response that was mechanistically independent of complement. In the present study, we investigated the function of CD59 in the MRL/lpr model of murine lupus. We backcrossed the Cd59a knockout (Cd59a(-/-)) mouse onto the MRL/lpr background and compared Cd59a(+/+)-MRL/lpr and Cd59a(-/-)-MRL/lpr littermates for the development of systemic autoimmunity. We found that CD59a deficiency significantly exacerbated the skin disease and lymphoproliferation characteristic of MRL/lpr mice. It also increased autoantibody titers and caused a higher level of proteinuria in male MRL/lpr mice. Bone marrow transfer experiments indicated that CD59a expression on both bone marrow-derived cells and peripheral tissues played a role in lymphoproliferation, whereas the skin disease phenotype is determined mainly by local CD59a expression. Importantly, C3 gene deletion or C5 neutralization with a blocking mAb in Cd59a(-/-)-MRL/lpr mice did not rescue the proautoimmune phenotype associated with CD59a deficiency. These results together suggest that CD59a inhibits systemic autoimmunity in MRL/lpr mice through a complement-independent mechanism.

Prison Break: Pathogens' Strategies To Egress from Host Cells

A wide spectrum of pathogenic bacteria and protozoa has adapted to an intracellular life-style, which presents several advantages, including accessibility to host cell metabolites and protection from the host immune system. Intracellular pathogens have developed strategies to enter and exit their host cells while optimizing survival and replication, progression through the life cycle, and transmission. Over the last decades, research has focused primarily on entry, while the exit process has suffered from neglect. However, pathogen exit is of fundamental importance because of its intimate association with dissemination, transmission, and inflammation. Hence, to fully understand virulence mechanisms of intracellular pathogens at cellular and systemic levels, it is essential to consider exit mechanisms to be a key step in infection. Exit from the host cell was initially viewed as a passive process, driven mainly by physical stress as a consequence of the explosive replication of the pathogen. It is now recognized as a complex, strategic process termed "egress," which is just as well orchestrated and temporally defined as entry into the host and relies on a dynamic interplay between host and pathogen factors. This review compares egress strategies of bacteria, pathogenic yeast, and kinetoplastid and apicomplexan parasites. Emphasis is given to recent advances in the biology of egress in mycobacteria and apicomplexans.

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.