Interior modification of Macrobrachium rosenbergii nodavirus-like particle enhances encapsulation of VP37-dsRNA against shrimp white spot syndrome infection.

BACKGROUND: Application of a virus-like particle (VLP) as a nanocontainer to encapsulate double stranded (ds)RNA to control viral infection in shrimp aquaculture has been extensively reported. In this study, we aimed at improving VLP's encapsulation efficiency which should lead to a superior fighting weapon with disastrous viruses. RESULTS: We constructed 2 variants of chimeric Macrobrachium rosenbergii nodavirus (MrNV)-like particles (V1- and V2-MrN-VLPs) and tested their efficiency to encapsulate VP37 double stranded RNA as well as WSSV protection in P. vannamei. Two types of short peptides, RNA-binding domain (RBD) and deca-arginine (10R) were successfully engineered into the interior surface of VLP, the site where the contact with VP37-dsRNA occurs. TEM and dynamic light scattering (DLS) analyses revealed that the chimeric VLPs remained their assembling property to be an icosahedral symmetric particle with a diameter of about 30 nm, similar to the original MrN-VLP particle. The superior encapsulation efficiency of VP37-dsRNA into V2-MrN-VLP was achieved, which was slightly better than that of V1-MrN-VLP but far better (1.4-fold) than its parental V0-MrN-VLP which the mole ratio of 7.5-10.5 for all VLP variants. The protection effect against challenging WSSV (as gauged from the level of VP37 gene and the remaining viral copy number in shrimp) was significantly improved in both V1- and V2-MrN-VLP compared with an original V0-MrN-VLP template. CONCLUSION: MrN-VLP (V0-) were re-engineered interiorly with RBD (V1-) and 10R (V2-) peptides which had an improved VP37-dsRNA encapsulation capability. The protection effect against WSSV infection through shrimp administration with dsRNA + V1-/V2-MrN VLPs was experimentally evident.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model.

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6-24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.

Infectious hypodermal and hematopoietic necrosis virus-like particle (IHHNV-VLP) induces peroxiredoxin expression and activity in Fenneropenaeus merguiensis.

Virus like particles (VLPs) are non-infectious nanoparticles containing repetitive, high density viral epitopes on the surface and can prevent viral infections in aquatic animals. Here, we evaluated the immuno-stimulation effect of infectious hypodermal and hematopoietic necrosis virus like particle (IHHNV-VLP) using a next generation sequencing in Fenneropenaeus merguiensis to identify the important immune-related genes that may prevent viral infection. The in situ target of IHHNV was predominantly found in gill tissue following IHHNV-VLP administration in juvenile shrimp. Comparative transcriptome analysis in the injected gills showed that there were 326 unigenes expressed differently than the mock-injected samples. One of the most differential genes between the two animal groups was the antioxidative gene, peroxiredoxin (FmPrx), that was up-regulated after 6 h post-VLP injection. Phylogenetic tree analysis showed that this gene could be found among many shrimp species and was closely clustered among Prx families. The expression of FmPrx was also detected in all tissues examined, thus suggesting the multi-functional roles of this gene in many tissues. Administration of IHHNV-VLP in vivo led to a significant increase in peroxidase activity in gill tissue-approximately two-fold versus control animals; the WSSV copy number was significantly reduced. These data suggest that IHHNV-VLP exerts an immune-stimulating effect by enhancing the level of immune-related genes including FmPrx and its corresponding peroxidase activity, which are a well-known part of the shrimp innate immune system.

Anatomical variants identified on chest computed tomography of 1000+ COVID-19 patients from an open-access dataset.

Chest computed tomography (CT) has been the preferred imaging modality during the pandemic owing to its sensitivity in detecting COVID-19 infections. Recently, a large number of COVID-19 imaging datasets have been deposited in public databases, leading to rapid advances in COVID-19 research. However, the application of these datasets beyond COVID-19-related research has been little explored. The authors believe that they could be used in anatomical research to elucidate the link between anatomy and disease and to study disease-related alterations to normal anatomy. Therefore, the present study was designed to investigate the prevalence of six well-known anatomical variants in the thorax using open-access CT images obtained from over 1000 Iranian COVID-19 patients aged between 6 and 89 years (60.9% male and 39.1% female). In brief, we found that the azygos lobe, tracheal bronchus, and cardiac bronchus were present in 0.8%, 0.2%, and 0% of the patients, respectively. Variations of the sternum, including sternal foramen, episternal ossicles, and sternalis muscle, were observed in 9.6%, 2.9%, and 1.5%, respectively. We believe anatomists could benefit from using open-access datasets as raw materials for research because these datasets are freely accessible and are abundant, though further research is needed to evaluate the uses of other datasets from different body regions and imaging modalities. Radiologists should also be aware of these common anatomical variants when examining lung CTs, especially since the use of this imaging modality has increased during the pandemic.

Mannosylated glycoconjugates on the surface of activated sperm in the giant freshwater prawn are crucial for sperm binding with the egg vitelline envelop.

The unusual morphology and poorly defined acrosome-like structure in the mature sperm of the giant freshwater prawn Macrobrachium rosenbergii has led to difficulties in identifying the state of sperm activation. Mature distal vas deferens sperm (dVSp) can be activated by the calcium ionophore A23187 to show acrosome reaction-like enzymatic activities that increase their binding and penetration capabilities. However, these short-lived enzymatic activities limit their usefulness as a marker of sperm activation for further qualitative and quantitative analyses, leading to our examining the alterations in the exposure of sperm surface glycoconjugates both as markers of sperm activation and for their role in gamete interaction. Our results showed that after A23187 treatment, there was an increased exposure of mannosylated glycoconjugates on the sperm surface revealed by significant Concanavalin A (Con A) staining. Furthermore, sodium metaperiodate pre-treatment, Con A pre-incubation, or co-incubation with α-mannose monosaccharides all significantly reduced A23187-induced dVSp binding to the egg vitelline envelop, demonstrating the importance of sperm surface mannosylated glycoconjugates in the binding process. These same pre-treatments of sperm also resulted in the inhibition of the binding of soluble vitelline envelop proteins (MrVE) to both the sperm surface and to mannosylated dVSp protein bands. Therefore, the present study demonstrated the importance of the exposure of mannosylated glycoconjugates on the surface of activated dVSp, both as a reliable marker of sperm activation and as a binding factor in the gamete interaction process. Furthermore, these findings allow for a better understanding of the surface glycoconjugate-mediated interaction process between gametes in this species of prawn.

Macrobrachium rosenbergii nodavirus virus-like particles attach to fucosylated glycans in the gills of the giant freshwater prawn.

The Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white-tail disease (WTD) in many species of shrimp and prawn, has been shown to infect hemocytes and tissues such as the gills and muscles. However, little is known about the host surface molecules to which MrNV attach to initiate infection. Therefore, the present study investigated the role of glycans as binding molecules for virus attachment in susceptible tissues such as the gills. We established that MrNV in their virus-like particle (MrNV-VLP) form exhibited strong binding to gill tissues and lysates, which was highly reduced by the glycan-reducing periodate and PNGase F. The broad, fucose-binding Aleuria Aurantia lectin (AAL) highly reduced MrNV-VLPs binding to gill tissue sections and lysates, and efficiently disrupted the specific interactions between the VLPs and gill glycoproteins. Furthermore, mass spectroscopy revealed the existence of unique fucosylated LacdiNAc-extended N-linked and O-linked glycans in the gill tissues, whereas beta-elimination experiments showed that MrNV-VLPs demonstrated a binding preference for N-glycans. Therefore, the results from this study highly suggested that MrNV-VLPs preferentially attach to fucosylated N-glycans in the susceptible gill tissues, and these findings could lead to the development of strategies that target virus-host surface glycan interactions to reduce MrNV infections.

Dual VP28 and VP37 dsRNA encapsulation in IHHNV virus-like particles enhances shrimp protection against white spot syndrome virus.

Accumulative evidence of using double stranded (ds) RNA encapsulated into virus like particle (VLP) nanocarrier has open feasibility to fight against shrimp viral infection in aquaculture field. In this study, we co-encapsulated VP37 and VP28 dsRNA into hypodermal and hematopoietic necrosis virus (IHHNV) like particle and investigated its protection against white spot syndrome virus (WSSV). Five micrograms of each dsRNA were used as starting materials to load into VLP, while the loading efficiency was slightly different, i.e, VP37 dsRNA had somewhat a better load into VLP's cavity. It was apparent that co-encapsulation of dual dsRNA showed a superior WSSV silencing ability than the single dsRNA counterpart as evidence by the lower WSSV gene expression and its copy number in the gill tissues. Besides, we also demonstrated that co-encapsulated dual dsRNA into IHHNV-VLP stimulated the increased number of hemocytes and the corresponding PO activity as well as up-regulated proPO gene expression in hemocytes to resist viral invasion after an acute stage of WSSV infection. This synergistic action of dual dsRNA encapsulated into IHHNV-VLPs could thus act to delay time of shrimp death and reduced shrimp cumulative mortality greater than the single, naked dsRNA treatment and positive control groups. The obtaining results would encourage the feasibility to use it as a new weapon to fight WSSV infection in shrimp aquaculture.

Existence and distribution of Niemann-Pick type 2C (NPC2) in prawn reproductive tract and its putative role as a cholesterol modulator during sperm transit in the vas deferens.

Sequestering of cholesterol (CHO) is a hallmark molecular event that is known to be associated with sperm gaining their fertilizing ability in a broad array of animals. We have shown previously that the level of CHO declines in the Macrobrachium rosenbergii sperm membrane when they are migrating into the vas deferens, prompting us to search for CHO transporters, one of which is Niemann-Pick type 2C (NPC2), within the prawn male reproductive tract. Sequence comparison of MrNPC2 with other NPC2, from crustaceans to mammals, revealed its conserved features in the hydrophobic cavity with 3 amino acids forming a CHO lid that is identical in all species analyzed. Expressions of MrNPC2 transcript and protein were detected in testicular supporting and interstitial cells and along the epithelial cells of the vas deferens. As confirmed by live cell staining, the testicular sperm (Tsp) surface was devoid of MrNPC2 but it first existed on the vas deferens sperm, suggesting its acquisition from the luminal fluid, possibly through trafficking of multi-lamellar vesicles during sperm transit in the vas deferens. We further showed that recombinant MrNPC2 had a high affinity towards CHO in the lipid extracts, either from Tsp or from lipid vesicles in the vas deferens. Together, our results indicated the presence of MrNPC2 in the male reproductive tract, which may play an important role as a CHO modulator between the sperm membrane and vas deferens epithelial communication.

Enhancement of shrimp health and immunity with diets supplemented with combined probiotics: application to Vibrio parahaemolyticus infections.

The application of probiotics for disease control in aquaculture is now a convincing approach towards replacement of antibiotics, which can cause adverse effects in aquatic animals and humans. In this study, we combined 2 probiotics, Lactobacillus acidophilus and Saccharomyces cerevisiae, with shrimp feed to create 2 formulas (WU8 and WU9), which were fed for 10 d to juvenile shrimp Penaeus vannamei. The shrimps were then subjected to a challenge infection with Vibrio parahaemolyticus, the causative agent of acute hepatopancreas necrosis disease (AHPND). The protective effects of probiotics against bacterial infection were investigated through histopathology of the hepatopancrease and immunological evaluation of shrimp. Both WU8 and WU9 probiotic mixtures (1:1, at 108 and 109 CFU kg diet-1) increased blasenzellen hepatopancreatic epithelial cells and reduced pathology caused by AHPND. After 10 d of feeding, hemocyte parameters, including the total hemocyte count, percent of granular hemocytes, and phenoloxidase activity, increased significantly and were still increasing at 24 h post infection. Crustin and penaeidin 3 genes were also highly upregulated in hemocytes before and after 24 h of bacterial challenge and significantly upregulated in the hepatopancreas 1 to 5 d post-infection. A significantly higher survival rate was observed in shrimp fed with the probiotic supplemented diet (>90%) in comparison to the control group (60%). In conclusion, probiotic mixtures of L. acidophilus and S. cerevisiae reduced hepatopancreas pathology and protected shrimp from a challenge with AHPND.

N-linked mannose glycoconjugates on shrimp thrombospondin, pmTSP-II, and their involvement in the sperm acrosome reaction.

Glycoconjugates in egg extracellular matrices are known to serve several functions in reproductive processes. Here, the presence of N-linked mannose (Man) glycoconjugates on shrimp thrombospondin ( pmTSP-II) and their physiological functions were investigated in the black tiger shrimp Penaeus monodon. A molecular analysis of pmTSP-II demonstrated anchorage sites for N-linked glycans in both the chitin-binding and TSP3 domains. The presence of Man residues was verified by concanavalin A lectin histochemistry on the purified fraction of pmTSP-II (250 kDa with protease inhibitor). The function of the Man glycoconjugates was evident by the Con A interference with the pmTSP-II-induced acrosome reaction (AR) as well as by the ability to recover the induction of the AR by the inclusion of Mans in the treatment mixture. In addition, the recombinant proteins of the three signature pmTSP-II domains expressed in E. coli (lacking glycosylation) and mannosidase-treated pmTSP-II showed a minimal ability to initiate the AR response. Together, these results provide evidence of the pivotal role that Man-linked pmTSP-II plays in modulating the shrimp sperm AR, a novel role for a TSP family protein in shrimp reproductive biology.

Hematopoietic tissue of Macrobrachium rosenbergii plays dual roles as a source of hemocyte hematopoiesis and as a defensive mechanism against Macrobrachium rosenbergii nodavirus infection.

White tail disease caused by Macrobrachium rosenbergii nodavirus (MrNV) infection takes place only in nauplii, not adults, of M. rosenbergii prawn. Hemocyte homeostasis and immune-related functions derived from the hematopoietic tissue (Hpt) in adult prawn are presumed to play roles in resisting viral infection. To elucidate the role of the Hpt cell response to MrNV, a comparative transcriptome analysis was performed with MrNV-infected prawn at various time intervals. The results showed that there were 462 unigenes that were differentially expressed between mock and infected samples. BlastX sequence analysis revealed that two proteins, crustacean hematopoietic factor (CHF) and cell growth-regulating zinc finger protein (Lyar), are involved in hemocyte hematopoiesis and are up-regulated during MrNV infection. In fact, genes involved in cell growth regulation and immunity were highly expressed at 6 h and decreased within 24 h post-infection. Localization studies in the Hpt tissue revealed the presence of anti-lipopolysaccharide factor (ALF) and CHF mRNAs in Hpt cells. Considering these findings, we concluded that resistance to MrNV infection in adult prawn is due to an increase in humoral immune factors and the acceleration of hemocyte homeostasis by the dual roles of the Hpt organ in M. rosenbergii.

The development of the cloaca in the human embryo.

Subdivision of cloaca into urogenital and anorectal passages has remained controversial because of disagreements about the identity and role of the septum developing between both passages. This study aimed to clarify the development of the cloaca using a quantitative 3D morphological approach in human embryos of 4-10 post-fertilisation weeks. Embryos were visualised with Amira 3D-reconstruction and Cinema 4D-remodelling software. Distances between landmarks were computed with Amira3D software. Our main finding was a pronounced difference in growth between rapidly expanding central and ventral parts, and slowly or non-growing cranial and dorsal parts. The entrance of the Wolffian duct into the cloaca proved a stable landmark that remained linked to the position of vertebra S3. Suppressed growth in the cranial cloaca resulted in an apparent craniodorsal migration of the entrance of the Wolffian duct, while suppressed growth in the dorsal cloaca changed the entrance of the hindgut from cranial to dorsal on the cloaca. Transformation of this 'end-to-end' into an 'end-to-side' junction produced temporary 'lateral (Rathke's) folds'. The persistent difference in dorsoventral growth straightened the embryonic caudal body axis and concomitantly extended the frontally oriented 'urorectal (Tourneux's) septum' caudally between the ventral urogenital and dorsal anorectal parts of the cloaca. The dorsoventral growth difference also divided the cloacal membrane into a well-developed ventral urethral plate and a thin dorsal cloacal membrane proper, which ruptured at 6.5 weeks. The expansion of the pericloacal mesenchyme followed the dorsoventral growth difference and produced the genital tubercle. Dysregulation of dorsal cloacal development is probably an important cause of anorectal malformations: too little regressive development may result in anorectal agenesis, and too much regression in stenosis or atresia of the remaining part of the dorsal cloaca.

Alpha-2 macroglobulin as a region-specific secretory protein in male reproductive tract, and its dynamics during sperm transit toward the female spermatheca in the blue crab.

A 250-kDa protein was isolated from fluid in the middle spermatic duct (MSD) of the blue crab (Portunus pelagicus). N-terminal and partial amino acid sequences revealed that this MSD-specific protein is highly similar to the plasma-enriched protein Alpha-2 macroglobulin (α2M). The P. pelagicus ortholog (Ppα2M) is a large glycoprotein possessing mannose and N-acetylglucosamine residues. Ppa2m mRNA was detected in the spermatic duct, androgenic gland, and hematopoietic tissue, whereas the protein was primarily observed in the apical cytoplasm of MSD epithelium and in the matrix of the acrosome of MSD sperm; distally within spermatic duct, Ppα2M was lost from the sperm membrane but remained in the sperm acrosome. These results suggest that Ppα2M is expressed and glycosylated in the epithelium of spermatic ducts, secreted into MSD fluid, taken up by sperm in the MSD, and removed from the surface of sperm during its transit towards the female spermatheca. Given that Ppα2M also exhibits protease inhibitor activity, we hypothesize that acrosome localized Ppα2M may suppress premature acrosome reaction during post-testicular sperm maturation in this crab.

Potential of RNAi applications to control viral diseases of farmed shrimp.

Viral pathogens pose a primary threat to global shrimp aquaculture. Despite the urgent industry need for them, practical anti-viral control methods are unavailable due, in part, to lack of an adaptive immune response in crustaceans that renders conventional vaccination methods ineffective. One currently studied method of high interest for protecting shrimp against viral infection relies on the post-transcriptional gene silencing mechanism called RNA interference (RNAi) that is induced by gene-specific constructs of double stranded RNA (dsRNA). Although this approach was first described for successful protection of shrimp against white spot disease (WSD) by injecting dsRNA specific to genes of white spot syndrome virus (WSSV) into shrimp in the laboratory in 2005 no practical method for use of dsRNA in shrimp farms has been developed to date. The apparent bottleneck for farm-scale applications of RNAi-mediated viral control in shrimp aquaculture is the lack of simple and cost-effective delivery methods. This review summarizes recent studies on use and delivery of dsRNA to shrimp via injection and oral routes in hatcheries and on farms and it discusses the research directions that might lead to development of practical methods for applications with farmed shrimp. Oral delivery methods tested so far include use of dsRNA-expressing bacteria as a component of dry feed pellets or use of living brine shrimp (Artemia) pre-fed with dsRNA before they are fed to shrimp. Also tested have been dsRNA enclosed in nanocontainers including chitosan, liposomes and viral-like particles (VLP) before direct injection or use as components of feed pellets for hatchery or pond-reared shrimp.

Presence of gonadotropin-releasing hormone-like peptide in the central nervous system and reproductive organs of the male blue swimming crab, Portunus pelagicus, and its effect on spermatogenesis.

Our previous studies have demonstrated that lamprey gonadotropin-releasing hormone-III (lGnRH-III)-like peptide occurs in the central nervous system (CNS) of decapod crustaceans (Macrobrachium rosenbergii, Penaeus monodon, Portunus pelagicus), and that lGnRH-III is the most potent in stimulating ovarian maturation compared with other GnRH isoforms. In this study, we examined the localization of lGnRH-III-like peptide in the CNS and male reproductive organs of the blue swimming crab by using anti-lGnRH-III as a probe. In the brain, lGnRH-III immunoreactivity (-ir) was detected in neurons of clusters 6, 10, 11, 14/15, 16, and 17 and in many neuropils. In the subesophageal ganglion, lGnRH-III-ir was present in neurons of the dorso-lateral and ventro-medial clusters. In the thoracic ganglia, lGnRH-III-ir was observed in the large-sized neurons between the thoracic neuropils and in the ventromedial cluster of the abdominal ganglia. In the testis, lGnRH-III-ir was detected in nurse cells, hemocytes, spermatids 2, and the outer and inner zones of the acrosomes of spermatozoa. Bioassay showed that lGnRH-III significantly increased the testis-somatic index, the percentage of late stages of seminiferous tubules (stages VII-IX), the diameter of the seminiferous tubules, and the number of BrdU-labeled early germ cells compared with the control groups. Thus, lGnRH-III-like peptide exists in the male crab and possibly enhances germ cell proliferation and maturation in the testes, leading to increased sperm production.

Expression of Penaeus monodon ortholog of Niemann-Pick type C-2 in the spermatic tract, and its role in sperm cholesterol removal.

Protein and lipid composition of sperm plasma membrane are modified as these gametes continue to mature during their transit along the spermatic tract. Our previous study revealed that during its journey through the spermatic duct of the black tiger prawn, Penaeus monodon, sperm cholesterol content decreases through the action of lipid-binding proteins within the luminal environment. In this study, the full cDNA sequence of epididymal secretory protein E1 (HE1), or Niemann-Pick C2 (NPC2), was cloned from P. monodon (termed Pmnpc2), and its conserved cholesterol/lipid-binding domain was characterized. The putative tertiary structure of PmNPC2 showed high similarity with the structure of Bos taurus NPC2. Pmnpc2 is expressed in many tissues, including the spermatic tract (i.e., testis, vas deferens, terminal ampoule) and the female thelycum. In situ hybridization revealed the presence of Pmnpc2 transcripts in the vas deferens, terminal ampoule, and thelycum epithelia, suggesting that PmNPC2 could be secreted into the lumen of the spermatic duct. A recombinant hexahistidine-tagged PmNPC2 (rPmNPC2-6His) was able to bind cholesterol and sperm lipid extracts, while co-incubation of sperm from the vas deferens with rPmNPC2-6His resulted in the depletion of cholesterol from these gametes. Together, these results suggest that PmNPC2 participates in sperm cholesterol efflux during the sperm maturation process in P. monodon. Mol. Reprod. Dev. 83: 259-270, 2016. © 2016 Wiley Periodicals, Inc.

Enhancement of trypsin-like enzymes by A23187 ionophore is crucial for sperm penetration through the egg vestment of the giant freshwater prawn.

We report the presence of trypsin-like enzymes preferring Boc-QAR-MCA substrate in sperm collected from different portions of male reproductive tracts of the giant freshwater prawn, Macrobrachium rosenbergii and compare enzyme activities before and after an A23187 calcium ionophore treatment. Fluorogenic enzyme assays revealed that testicular sperm lysates showed high trypsin-like enzyme activity but the activity was relatively low in vas deferens sperm lysates as well as in the live sperm. Upon sperm treatment with A23187, trypsin-like activity was greatly enhanced in distal vas deferens sperm. Substrate- and inhibitor-based localization studies indicated that the sperm trypsin-like enzymes were not of a soluble type but were rather of a membrane-borne type, localized at the anterior spike and upper part of the main body. Notable structural changes were also evident in A23187-induced sperm including extensive ruffling of the sperm membrane structure at the base of the main body thereby supporting the acrosome reaction response in this species. We further proved by substrate inhibition assays that the enhanced trypsin-like enzyme activity participates in sperm penetration through the vitelline envelope, a novel sperm-egg penetration mechanism that is unique in this species.

Lectin-Based Profiling of Coelomocytes in Holothuria scabra and Expression of Superoxide Dismutase in Purified Coelomocytes.

Coelomocytes are the first line of immune defense in marine animals. Their distributions are greatly variable even in the close animal species. In this study, we used lectin staining to aid in the classification and purification of these cells for further investigation of SOD distribution among coelomocytes of H. scraba. We classified coelomocytes into four types: type 1, lymphocytes; type 2, phagocytes; type 3, spherulocytes; and type 4, giant cells. Among four lectins used, Con A appeared to give a broad reactivity against most coelomocytes, except for giant cells. In addition, phagocytes usually engaged the highest fluorescent intensity with most lectins, with the exception of PNA, for which spherulocytes possessed the highest fluorescent intensity. Using FACS for fraction collection, it was found that F1 fraction contained the purest phagocyte population (> 95%), which was highly reactive with anti- superoxide dismutase (SOD) as revealed by immunoblotting and immunofluorescence staining, although some minor staining was also detected in spherulocytes. Our results thus provide a fundamental platform for comparing alterations that may happen to the population and SOD contents of coelomocytes when the sea cucumber is subjected to environmental changes that would activate their immune responses.

A role of oestrogen in aggravating SLE-like syndrome in C4-deficient mice.

BACKGROUND: As one of the epigenetic factors, oestrogen is considered to be a predisposing factor that is associated with a susceptibility to autoimmune disease development in women including systemic lupus erythematosus (SLE). Here, we proposed that oestrogen is also imparted in a post-lupus symptomatic enhancement as studied in the C4-deficient (C4-/-) mice model known to develop SLE-like symptoms. METHODS: Fifty-six C4 knockout mice were ovariectomised (OVX) to eliminate the effect of endogenous feminine hormones followed by 17-ß oestradiol (E2) administration in both dose- and time-dependent manners. Histopathological features of kidneys and spleens were studied by histological and immunofluorescent staining. The relative expression levels of IgG and IgM were measured densitometrically on their immunoreactive bands and the level of IgG-anti-double stranded (ds) DNA was measured by ELISA. RESULTS: E2-treated mice displayed a gradual increase in immune complex deposition (both IgG and IgM) in glomeruli and proximal convoluted tubules. An increased reactivity of autoantibodies against dsDNA correlated with increasing doses and longer exposure to E2 treatments. In addition, enlargement of the spleen (splenomegaly) was also observed in E2-treated mice. CONCLUSIONS: Our results support the hypothesis that oestrogen aggravates severity of the SLE-like symptoms in C4-deficient mice.