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C-type lectin receptors, as the important members of pattern-recognition receptors, play the crucial roles in the innate immune system, which discriminate self and non-self by recognizing and binding the carbohydrates on the surface of microorganism. In this study, we identified a C-type lectin receptor gene in Qihe crucian carp Carassius auratus (named as CaCLR). The full-length cDNA of CaCLR was composed of 1130 bp, with a 226 bp 5'-untranslated region (UTR), a 792 bp ORF encoding a 263aa protein, and a 112 bp 3'-UTR with a polyadenylation signal sequence AATAAA and a poly (A) tail. The predicted amino acid sequence of CaCLR is a single transmembrane receptor with a typical carbohydrate recognition domain (CRD) at its C-terminus. With regard to the mRNA transcript of CaCLR, it was ubiquitously detected in the tested tissues, among which it was the most abundant in head kidney. The temporal expressions of CaCLR were obviously up-regulated in liver, spleen, kidney, and head kidney after Aeromonas hydrophila and poly I: C challenge, respectively, and the patterns of expression changes were in a time-depended manner. The recombinant CaCLR (rCaCLR) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with lipopolysaccharide (LPS), peptidoglycan (PGN), β-Glucan, and Mannan, as well as five microorganisms including fungus (Saccharomyces cerevisiae), Gram-negative bacteria (A. hydrophila, E. coli and Vibrio anguillarum), and Gram-positive bacteria (Micrococcus lysodeikticus). In the presence of rCaCLR, the eliminating capacity against A. hydrophila could be enhanced in C. auratus. Taken together, CaCLR is involved in the antibacterial defense in C. auratus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required., Competing Interests: Declaration of Competing Interest None., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defences of various invertebrates. Whether or not laccase exists in shrimp and its function is still poorly understood. In this study, a laccase (LvLac) was cloned and identified from Litopenaeus vannamei for the first time. The full length of LvLac is 3406 bp, including a 2034 bp open reading frame (ORF) coding for a putative protein of 677 amino acids with a signal peptide of 33 aa. LvLac contains three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine, respectively. Phylogenetic analysis revealed that LvLac was close to insects laccase 1 family. LvLac expression was most abundant in heart and the crude LvLac protein could catalyze the oxidation of hydroquinone. Real-time PCR showed that LvLac expression was responsive to Vibrio parahaemolyticus, Micrococcus lysodeikticus and white spot syndrome virus (WSSV) infection. Knockdown of LvLac enhanced the sensitivity of shrimps to V. parahaemolyticus and M. lysodeikticus challenge, suggesting that LvLac may play a positive role against bacterial pathogens., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lysozymes are important defense proteins of the innate immune system and possess high antibacterial activities. In the present study, a full-length c-type lysozyme cDNA (HtLysC) was cloned and characterized from taimen (Hucho taimen, Pallas). The cDNA contains an open reading frame (ORF) of 432 bp encoding 143 amino acid (aa), with 97% identity to LysC of Rainbow trout (Oncorhynchus mykiss). The amino acid sequence possessed a LYZ1 domain (16-140 aa) which contained two conserved residues (Glu 50 and Asp 67), eight conserved cysteine residues and a calcium binding site. RT-PCR analysis showed that HtLysC transcripts were most abundant in liver and less in muscle. The expression of HtLysC was up-regulated in the liver when challenged with Yersinia ruckeri. The recombinant HtLysC (rHtLysC) had lytic activities against Micrococcus lysodeikticus, Aeromonas salmonicida and Y. ruckeri. Enzyme assay showed that the optimal temperature and pH of rHtLysC were 55 °C and 6.0, respectively. Taken together, these results indicated that HtLysC might play an important role in innate immune defense against bacterial pathogens as a functional lysozyme., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The Toll and immune deficiency (IMD) pathways are essential for inducing immune related genes during invasion of pathogens. In the present study, transcripts of eight pathway-related genes in Litopenaeus vannamei, including Toll, IMD, Pelle, IAP1, TRAF6, ALF, Crustin and Penaeidin3 were analyzed to further understand the potential relationship between Toll and IMD pathway. The high transcription levels of TRAF6, Pelle, Toll, IMD and IAP1 in selected tissues indicates their functional roles in Toll and IMD pathways. The increased mRNA expression of Toll and IMD detected in the early stage might suggest the inducible role of Toll and IMD upon bacterial infection. Moreover, the continuous increase of IMD and the high level of Pelle and TRAF6 in Vibrio anguillarum challenged group indicated that Gram-negative bacterium can activate both the Toll and IMD signaling pathway. Silencing of Toll by a dsRNA-mediated RNAi strongly increased the transcripts of IMD, Pelle, TRAF6, IAP1 and Akirin, knocking down of IMD also markedly increased the transcripts of Toll, Pelle, IAP1 and Akirin. Furthermore, ALF expression was significantly increased in response to V. anguillarum and Micrococcus lysodeikticus challenge, while the transcripts of Crustin and Pen3 in hemocytes were significantly reduced in V. anguillarum group, but rose significantly following M. lysodeikticus infection. In summary, we speculate that Toll and IMD pathway are not independent in shrimp, but linked to defense against bacterial infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Serine protease inhibitors (serpins) are widely known to its inhibitory role on proteases involved in the immune responses. Herein, a novel serine protease inhibitor (Lvserpin7), encoding for 411 amino acids with calculated molecular mass of 46.29 kDa and isoelectric point of 6.98 was characterized from the Pacific white shrimp Litopenaeus vannamei. Lvserpin7 shared 92.9% identities to Penaeus monodon serpin7. Among the tested tissues, Lvserpin7 was mainly expressed in hemocytes and gill. The expression profiles analysis indicated that Lvserpin7 was significantly up-regulated in the early stage upon Vibrio anguillarum, Micrococcus lysodeikticus or White Spot Syndrome Virus (WSSV) infection. Fusion protein expression was induced by IPTG, and the purified recombinant Lvserpin7 protein (rLvserpin7) binds to both the Gram-positive and Gram-negative bacteria. Also rLvserpin7 exhibited inhibitory activity against the proteases secreted by Bacillus subtilis. Moreover, rLvserpin7 showed inhibition role on prophenoloxidase activation. To recap, we proposed that Lvserpin7 was implicated in the shrimp immunity via the inhibition of bacterial proteases and proteases involved in prophenoloxidase system., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anti-lipopolysaccharide factor (ALF) is one of the widely-studied antimicrobial peptides (AMPs) with broad-spectrum antibacterial activity and antiviral property. Previous studies show the existence of multiform of ALFs in crustacean which are important for immunity of the animals. In the present study, we characterized one isoform of ALF from the Chinese shrimp Fenneropenaeus chinensis (FcALF2). Tissue distribution analysis revealed that FcALF2 showed the highest expression level in the lymphoid organ (Oka) of the shrimp. The expression level of FcALF2 in shrimp was significantly up-regulated when they were injected with Micrococcus lysodeikticus and Vibrio anguillarum. A peptide corresponding to the LPS-binding domain of FcALF2 (FcALF2-LBD) was synthesized to analyze its antimicrobial activities. Data demonstrated that FcALF2-LBD possessed strong antibacterial activity against Gram-positive bacteria Micrococcus luteus and M.lysodeikticus with MIC ranges of 2-4 μM and 1-2 μM respectively and significant inhibition activity against white spot syndrome virus (WSSV). The antibacterial activities of the sequence modified peptides (FcALF2-LBDb, FcALF2-LBDv) were apparently enhanced and broadened after the amount of basic amino acids was increased in the synthetic LPS-binding domain. These data provide more insights into understanding the function of LPS-binding domain of ALF and the role of ALF in shrimp immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Calnexin (Cnx) is an endoplasmic reticulum membrane-bound lectin chaperone that comprises a dedicated maturation system with another lectin chaperone calreticulin (Crt). This maturation system is known as the Cnx/Crt cycle. The main functions of Cnx are Ca(2+) storage, glycoprotein folding, and quality control of synthesis. Recent studies have shown that Cnx is important in phagocytosis and in optimizing dendritic cell immunity. However, the functions of Cnx in invertebrate innate immunity remain unclear. In this research, we characterized Cnx in the kuruma shrimp Marsupenaeus japonicus (designated as MjCnx) and detected its function in shrimp immunity. The expression of MjCnx was upregulated in several tissues challenged with Vibrio anguillarum. Recombinant MjCnx could bind to bacteria by binding polysaccharides. MjCnx protein existed in the cytoplasm and on the membrane of hemocytes and was upregulated by bacterial challenge. The recombinant MjCnx enhanced the clearance of V. anguillarum in vivo, and the clearance effects were impaired after silencing MjCnx with RNA interference assay. Recombinant MjCnx promoted phagocytosis efficiency of hemocytes. These results suggest that MjCnx functions as one of the pattern recognition receptors and has crucial functions in shrimp antibacterial immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Freshwater snail Physa acuta has been considered as an important invasive species and medical mollusc. Field investigation has shown that this snail could survive better than other snails in polluted water bodies. To understand the immune mechanisms of P. acuta, suppression subtractive hybridization hepatopancreas cDNA library has been constructed with bacterial challenge. In this study, a full-length cDNA of a novel goose-type lysozyme (PALysG) has been identified from P. acuta by EST and RACE technique. The conservative structure domains share high homology with other molluscan g-type lysozymes including the SLT domain, the substrate binding sites, the catalytic residues, three alpha-helices structures and six molluscan specific cysteines. Meanwhile, PALysG is the first record of goose-type lysozyme in Gastropoda. Real-time PCR indicated that PALysG mRNA had been expressed significantly at high levels in hepatopancreas for 8-48 h. PALysG recombinant protein displayed the lytic activity of g-type lysozyme with other organisms against Micrococcus lysodikicus., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

NF-κB dependent antimicrobial peptides (AMPs) are of critical importance in protecting insects or mammals from microorganisms infection. However, we still do not make clear signaling pathways in regulating AMPs expression in shrimps. In this study, RNAi approach was used to study differences between Toll signaling pathway and immune deficiency signaling pathway in regulating the transcription of NF-κB dependent AMPs post bacteria challenge. Results showed that the transcription level of anti-lipopolysaccharide factor was highly suppressed in Litopenaeus vannamei immune deficiency (LvIMD) silenced shrimps by gene specific dsRNA compared to Litopenaeus vannamei Toll (LvToll) silenced shrimps with or without Vibrio anguillarum and Micrococcus lysodeikticus challenge. Conversely the transcription level of penaeidin3a was significantly suppressed in LvToll silenced shrimps compared to LvIMD silenced shrimps. However, no obvious difference was found in regulating the transcription of CrustinP. Meanwhile, we found that silencing LvToll both down regulated the transcription of Dorsal and Relish while silencing LvIMD only down regulated the transcription of Relish. At last, shrimp survival experiment showed that post V. anguillarum challenge high mortality was found both in LvToll and LvIMD silenced groups while post M. lysodeikticus challenge we saw high mortality only in LvToll silenced group. Hence, we conclude that shrimp L. vannamei Toll pathway and IMD pathway might be different in regulating the transcription of NF-κB dependent AMPs and responding to bacteria challenge but not independent of each other., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

As important arthropod immune responses, prophenoloxidase (proPO) activation and Toll pathway initiation are mediated by serine proteinase cascades and regulated by serpins. Herein, a serine protease inhibitor (Lvserpin), encoding for 415 amino acids with calculated molecular weight of 46,639 Da and isoelectric point of 7.03 was characterized from the Pacific white shrimp Litopenaeus vannamei. Multiple sequence alignment revealed that Lvserpin shared the highest similarity with Penaeus monodon serpin6 (87%). Quantitative real-time PCR (qRT-PCR) results showed that the transcripts of Lvserpin were detected in all the examined tissues and most highly expressed in gill. The expression profiles of Lvserpin were greatly fluctuated upon infection of Vibrio anguillarum, Micrococcus lysoleikticus or White Spot Syndrome Virus (WSSV). Double stranded RNA-mediated suppression of Lvserpin resulted in a significant increase in the transcripts of two clip-domain serine proteinases (PPAE and PPAF), prophenoloxidase (proPO), anti-lipopolysaccharide factor (ALF), Crustin and penaeidin3 (Pens3) and also increased the high cumulative mortality post V. anguillarum injection. Besides, the recombinant Lvserpin protein (rLvserpin) was purified and exhibited inhibitory activity against trypsin. Also the rLvserpin showed inhibition on prophenoloxidase activation and bacterial growth. Hence, we proposed that the Lvserpin played important role in the shrimp innate immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The objectives of this study were: 1) to investigate whether recombinant channel catfish lysozyme-g (CC-Lys-g) produced in Escherichia coli expression system possesses any lysozyme activity; and 2) to evaluate whether channel catfish lysozyme-g plasmid DNA could be used as an immunostimulant to protect channel catfish against Aeromonas hydrophila infection. Recombinant CC-Lys-g produced in E. coli expression system exhibited significant (P < 0.05) lytic activity against Gram-positive Micrococcus lysodeikticus and Gram-negative A. hydrophila. When pcDNA3.2-vectored recombinant channel catfish lysozyme-g (pcDNA-Lys-g) was transfected in channel catfish gill cells G1B, the over-expression of pcDNA-Lys-g offered significant (P < 0.05) protection to G1B cells against A. hydrophila infection. When channel catfish were intraperitoneally injected with pcDNA-Lys-g along with an adjuvant QCDCR, the transcriptional level of Lys-g was significantly (P < 0.05) increased. When pcDNA-Lys-g injected fish was challenged with a highly virulent A. hydrophila strain AL-09-71, pcDNA-Lys-g offered 100% protection to channel catfish at two days post DNA injection. Macrophages of fish injected with pcDNA-Lys-g produced significantly (P < 0.05) higher amounts of reactive oxygen species and nitric oxide than that of fish injected with pcDNA vector alone at two days post DNA injection. Taken together, our results suggest that pcDNA-Lys-g could be used as a novel immunostimulant to offer immediate protection to channel catfish against A. hydrophila infection., (Published by Elsevier Ltd.)

Understanding the outcomes of host-parasite interactions in nature is in high demand as parasites and pathogens are important for several ecological and evolutionary processes. Ecological immunology (ecoimmunology) has a key role in reaching this goal because immune defence is the main physiological barrier against infections. To date, ecoimmunological studies largely lean on measuring constitutive immune defences (components of defence that are always active). However, understanding the role of inducible components of immune function is important as the immune system is largely an inducible defence. Measuring such defences can be complicated as different parasites may activate different immune cascades, and expression of different immune traits may not be independent. We examined the suitability of different immune activation techniques for the freshwater snail Lymnaea stagnalis. By experimentally challenging snails with different immune elicitors [injection with snail saline (i.e. wounding), lyophilized Escherichia coli cells, lyophilized Micrococcus lysodeikticus cells, healthy snail gonad, and trematode-infected snail gonad; maintenance in microorganism-enriched water] and measuring phenoloxidase-like and antibacterial activity of their haemolymph, we found increased immune activity against some immune elicitors, but also decreased activity. Our findings suggest potentially complicated relationships among immune traits, and propose suitable techniques for ecological studies in this study system.

Insect prophenoloxidase (PPO) is essential for physiological functions such as melanization of invading pathogens, wound healing and cuticle sclerotization. The insect PPO activation pathway is well understood. However, it is not very clear how PPO is released from hemocytes and how PPO takes part in cellular immunity. To begin to assess this, three Drosophila melanogaster PPO genes were separately fused with GFP at the C-terminus (rPPO-GFP) and were over-expressed in S2 cells. The results of staining and morphological observation show that rPPO-GFP expressed in S2 cells has green fluorescence and enzyme activity if Cu(2+) was added during transfection. Each rPPO-GFP has similar properties as the corresponding rPPO. However, cells with rPPO-GFP over-expressed are easier to trace without PO activation and staining. Further experiments show that rPPO1-GFP is cleaved and activated by Drosophila serine protease, and rPPO1-GFP binds to Micrococcus luteus and Beauveria bassiana spores as silkworm plasma PPO. The above research indicates that the GFP-tag has no influence on the fusion enzyme activation and PPO-involved innate immunity action in vitro. Thus, rPPO-GFP may be a convenient tool for innate immunity study in the future if it can be expressed in vivo.

This study firstly reports the characterization of a functional IκB homologue, FcCactus in Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcCactus consists of a 1359 bp open reading frame (ORF) encoding a 453 amino acid protein with a predicted molecular weight (MW) of 48.46 kDa and theoretical pI of 5.23. Phylogenetic analysis and multiple alignments revealed that the deduced amino acid sequence of FcCactus cDNA had high similarities to Cactus or IκB reported in seven other arthropods. Genomic DNA sequence of FcCactus was also obtained with a length of more than 17698 bp and constituted of seven exons and six introns. Analysis on 5'-upstream regulatory region of its DNA sequence revealed that it contained the core promoter sequence with the TATA-box and transcription start site existing in it; furthermore, various transcription factor binding sites (HSF, Hb, BR-C Z, Dfd, CF2-II, Croc, Ttk, Dorsal, and c-Rel) were predicted. Spatial expression profiles showed that FcCactus mRNA had the highest expression level in muscle, hemocytes, heart and lymphoid organ. Gram-positive bacteria (Micrococcus lysodeikticus) and Gram-negative bacteria (Vibrio anguillarium) injection to shrimp caused the modulation of FcCactus at the transcription level. DsRNAi (double-strand RNA interference) approach was used to study the function of FcCactus and the data showed that FcCactus could regulate the expression of different antimicrobial peptides (AMPs) and antiviral factor (AV). The present data showed that FcCactus might play important roles in regulating the immune response of shrimp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The myeloid differentiation factor 88 (MyD88) is an important adapter protein which links members of the toll-like receptor (TLR) to the downstream components to activate related signaling pathways. In the present study, a MyD88 homolog (FcMyD88) was cloned from penaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88 consisted of 1434 bp encoding a polypeptide of 477 amino acids which contains a death domain (DD) and a typical TLR and interleukin-1 receptor (IL-1R)-related (TIR) domain. Homology analysis revealed that the predicted amino acid (aa) sequence of FcMyD88 shared high similarities with a variety of previously reported MyD88s. The time-dependent expression patterns of FcMyD88 in cephalothoraxes of shrimp injected with Vibrio anguillarum (Gram-negative bacteria, G(-)), Micrococcus lysodeikticu (Gram-positive bacteria, G(+)) and white syndrome spot virus (WSSV) were analyzed at transcription and protein level by real-time PCR and western blotting, respectively. The expression level of FcMyD88 mRNA was significantly up-regulated at one hour (h), 12 h and 24 h after stimulation with both V. anguillarum and M. lysodeikticu. The expression level of FcMyD88 protein was 2-fold up-regulated at 12 h post injection (hpi) of inactivated V. anguillarum while it didn't change after M. lysodeikticu injection during this period. After WSSV injection, the expression level of FcMyD88 mRNA remained relatively constant, while the FcMyD88 protein was significantly up-regulated at 12 and 24 hpi. These results suggested that the MyD88-dependent signaling pathway could be involved in the defense of both bacteria and WSSV infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A screening study of in vitro antibacterial activity was conducted in marine bivalves with economical importance and widespread along the coast of Galicia (NW Spain). Hemocyte lysate supernatant (HLS) and plasma of Mytilus galloprovincialis, Ostrea edulis, Crassostrea gigas, Ruditapes decussatus, Ruditapes philippinarum, and Cerastoderma edule were incubated with Vibrio splendidus and Micrococcus sp. HLS and plasma for all the species demonstrated antibacterial activity, and C. edule had the highest activity per unit of protein in these hemolymph fractions. Significant differences were not found between HLS and plasma activities. Furthermore, antibacterial activity against Micrococcus sp. (Gram-positive) was stronger than against V. splendidus (Gram-negative)., (Copyright © 2010 Elsevier Inc. All rights reserved.)

A crustin-like antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustin-like AMP from other shrimp crustins. The differential expression of the crustin-like AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two β-glucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two gram-positive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded pre- and post-challenge white spot syndrome virus (WSSV) by semi-quantitative RT-PCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustin-like AMP was found to be constitutively expressed in the animal and a significant down-regulation could be noted post-challenge WSSV. Remarkable down-regulation of the gene was observed in the immunostimulant fed animals pre-challenge followed by a significant up-regulation post-challenge WSSV. Tissue-wise expression of crustin-like AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustin-like AMP and confer significant protection to P. monodon against WSSV infection., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Rel/NFkappaB is a family of transcription factors. In the present study, a Rel/NFkappaB family member, Dorsal homolog (FcDorsal) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length cDNA of FcDorsal consists of 1627bp, revealed a 1071bp open reading frame encoding 357 aa. The predicted molecular weight (MW) of the deduced amino acid sequence of FcDorsal was 39.78kDa, and its theoretical pI was 8.85. Amino acid sequence analysis showed that FcDorsal contains a Rel homolog domain (RHD) and an IPT/TIG (Ig-like, plexins and transcriptions factors) domain. The signature sequence of dorsal protein existed in the deduced amino acid sequence. Spatial expression profiles showed that FcDorsal had the highest expression level in the hemocytes and lymphoid organ (Oka). The expression profiles in the hemocytes and lymphoid organ were apparently modulated when shrimp were stimulated by bacteria or WSSV. Both Gram-positive (G(+)) bacteria (Micrococcus lysodeikticus) and Gram-negative (G(-)) bacteria (Vibrio anguillarium) injection to shrimp caused the up-regulation of FcDorsal at the transcription level. DsRNA approach was used to study the function of FcDorsal and the data showed that FcDorsal was related to the transcription of Penaeidin 5 in shrimp. The present data provide clues that FcDorsal might play potential important roles in the innate immunity of shrimp. Through comparison of the expression profiles between FcDorsal and another identified Rel/NFkappaB member (FcRelish) in shrimp responsive to WSSV challenge, we speculate that FcDorsal and FcRelish might play different roles in shrimp immunity., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Bivalves are filter-feeders that can accumulate large numbers of bacteria, in particular Vibrio species; these can persist within bivalve tissues largely depending on their sensitivity to the hemolymph bactericidal activity. In this work, functional parameters of the hemolymph of Mytilus galloprovincialis were evaluated in response to in vivo challenge with different bacteria (Gram(-) Vibrio anguillarum and V. splendidus, Gram+ Micrococcus lysodeikticus). Mussels were injected with heat-killed bacteria or PBS-NaCl (controls) and hemolymph sampled from 3 to 48 h post-injection (p.i.). In hemocytes, all bacteria induced significant lysosomal membrane destabilisation (LMS) from 3 h p.i. with V. splendidus >V. anguillarum >M. lysodeikticus. LMS showed recovery for both M. lysodeikticus and V. anguillarum, whereas a further time-dependent decrease was observed for V. splendidus. Bacterial challenge also induced a rapid (from 3 h p.i.) and significant increase in serum lysozyme activity; the effect was persistent with M. lysodeikticus and transient for the two Vibrio species. In order to evaluate whether in vivo challenge may affect the subsequent capacity of hemolymph to kill bacteria, the bactericidal activity was tested in an in vitro assay towards E. coli. At 48 h. p.i. hemolymph samples from V. anguillarum-injected mussels showed a significant increase in E. coli killing (+35% with respect to controls); a smaller effect was observed with V. splendidus-injected mussels (+16%), whereas M. lysodeikticus was ineffective. Moreover, hemolymph from V. anguillarum-injected mussels showed an in vitro bactericidal activity towards V. anguillarum 2-folds higher than that of controls. Changes in total hemocyte counts (THC) and in hemocyte populations were evaluated by Flow cytometry at 6 and 48 h p.i., indicating a decrease in THC followed by recovery with all bacteria. Moreover, at 6 h p.i. a general decrease in the percentage of granulocytes was observed (V. splendidus >V. anguillarum >M. lysodeikticus), followed by complete and partial recovery with M. lysodeikticus and V. anguillarum, respectively, but not with V. splendidus. The results demonstrate the existence of differential functional immune responses in M. galloprovincialis to different bacteria.

Mussels are filter-feeders living in a bacteria-rich environment. We have previously found that numerous bacterial species are naturally present within the cell-free hemolymph, including several of the Vibrio genus, whereas the intra-cellular content of hemocytes was sterile. When bacteria were injected into the circulation of the mussel, the number of living intra-hemocyte bacteria dramatically increased in less than an hour, suggesting intense phagocytosis, then gradually decreased, with no viable bacteria remaining 12h post-injection for Micrococcus lysodeikticus, 24h for Vibrio splendidus and more than 48 h for Vibrio anguillarum. The total hemocyte count (THC) was dramatically lowered by the bacterial injections, as quantified by flow cytometry. V. splendidus induced the strongest decreases with -66% 9h post-injection of living bacteria and -56% 3h post-injection of heat-killed bacteria. Flow cytometry was used to identify three main sub-populations of hemocytes, namely hyalinocytes, small granulocytes and large granulocytes. When THC was minimal, i.e. within the first 9h post-injection, proportions of the three cell categories varied dramatically, suggesting differential involvement according to the targets, but small granulocytes remained the majority. According to a decrease in their number followed by an increase (+90% at 12h with living V. splendidus), hyalinocytes also appeared to be involved as cellular effectors of antibacterial immunity, despite possessing little capacity for phagocytosis and not containing antimicrobial peptides.

Defensin, mytilin and myticin are antimicrobial peptides (AMP) involved in mussel innate immunity. Their in vitro antibacterial activity is different according to the targeted bacterial species. To determine if this specificity is correlated to different regulations of gene expressions, adult mussels were challenged in vivo with either Vibrio splendidus LGP32, Vibrio anguillarum, Micrococcus lysodeikticus or by heat shock. RNAs were isolated from circulating hemocytes and AMP mRNAs were quantified by Q-PCR using 28S rRNA as housekeeping gene. In addition, HSP70 gene expression was also quantified as representing non-specific response to stress. In naïve mussels, the three AMP mRNAs were present in dramatically different quantities. Compared to defensin, myticin was expressed 300-fold more and mytilin 30-fold more. HSP70 was found expressed 80-fold more than defensin. AMP genes were differentially regulated according to the challenging bacteria, M. lysodeikticus being the only one inducing down-regulation. Such variations in mRNA quantities were observed immediately after challenging, lasting less than 24h. Only V. anguillarum effect was observed later, between 12h and 3 days post-challenge. Compared to their background expression in naïve mussels, the major effect of V. splendidus was the decrease of mytilin and myticin mRNAs, V. anguillarum mainly increased both mytilin and HSP70 mRNAs, whereas M. lysodeikticus almost suppressed defensin mRNA. As expected, heat shock increased HSP70 mRNA, but also myticin mRNA. Consequently, AMP genes responded specifically to the challenges, confirming that at least some of the innate immune mechanisms are specifically orientated.

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by antibodies to DNA (anti-DNA). Although these antibodies have features of antigen drive, the source of this DNA is not defined. To assess the potential role of foreign and self-DNA as driving antigens, the specificity of SLE sera for bacterial and mammalian DNA was evaluated. Micrococcus lysodeikticus (MC) and calf thymus (CT) DNA were tested as antigens, with absorption on CT DNA columns used to identify antibodies to antigenic sites on the two DNA. Among 9 sets of longitudinal sera tested, all showed binding to both DNA, and none showed exclusive or predominant binding to CT DNA. With absorbed sera, antibodies could be distinguished in terms of cross-reactive or selective binding to the DNAs. These findings suggest that anti-DNA antibodies vary in specificity and are consistent with a role of both foreign and self-DNA in anti-DNA induction.

Complete sequence of HSP70 cDNA from the mussel, Mytilus galloprovincialis was established before quantifying its expression following moderate heat shock or injection of heat-killed bacteria. HSP70 cDNA is comprised of 2378 bp including one ORF of 654 aa, with a predicted 70 bp 5'-UTR and a 343 bp 3'-UTR (GenBank, 18 Jan 05, AY861684). Alignment identity ranged from 89% for Crassostrea ariakensis to 72% for C. virginica. Curiously, HSP70 gene and cDNA sequences from M. galloprovincialis, deposited later (03 and 27 May), show only 73% identity with the present sequence. Meanwhile, characteristic motifs of the HSP70 family were located in conserved positions. Expression of HSP70 gene was quantified on circulating hemocyte mRNA using Q-PCR after RT using random hexaprimers. Housekeeping gene was 28S rRNA. Four stresses were applied: heat shock that consisted of immersing mussels for 90 min at 30 degrees C and returning them to 20 degrees C sea water, one injection of heat-killed Gram-negative bacteria, Vibrio splendidus LGP32, one injection of heat-killed Gram-negative bacteria Vibrio anguillarum, one injection of heat-killed Gram-positive bacteria Micrococcus lysodeikticus. We found no significant modification of 28S rRNA gene expression. Significant increase of 5.2 +/- 0.4 fold the ratio HSP70/28S rRNA was observed 6 h after heat shock and was maximum at 15 h (6.1 +/- 1.1), and still significant after 24 h (1.7 +/- 0.03). Similarly, injecting V. anguillarum resulted in a significant increase of 2.7 +/- 0.1 after 12 h. Expression was maximum after 48 h (5.2 +/- 0.05) and returned to baseline after 72 h. In contrast, injecting V. splendidus or M. lysodeikticus failed to significantly modulate HSP70 gene expression at least during the first 3 days post-injection. Consequently, mussel hemocytes appeared to discriminate between pathogenic and non-pathogenic Vibrios, as well as between Gram-negative and Gram-positive bacteria.

The notion that a trade-off exists between immunity and reproduction is now a central concept in theories of sexual selection. However, whether such a trade-off exists between immunity and gamete viability has not been established. Here we show that genetic variance for high levels of an immune response required to fight bacterial infections is associated with genetic variance for low sperm viability. These data have implications for our understanding of sexual selection mechanisms and of reproductive costs in male longevity.

This study investigated the potential in vivo immunotoxic effects of tributyltin (TBT) on amoebocytes of 6-armed seastar Leptasterias polaris. Tested animals were contaminated by trophic transfer via alive contaminated prey consisting of blue mussels (3microg TBT g(-1) wet weight (WW) tissue) exposed to seawater containing dissolved TBT. Four biomarkers of immunotoxicological effects were monitored over 45 days at different sampling times (9, 24, 48 and 72h, 11, 18, 25, 32 and 45 days): amoebocytes count (AC), cell viability using Trypan blue exclusion test, phagocytic activity (PA) using a suspension of dead bacteria labelled with fluorescein isothiocyanate (FITC) and injected directly in the coelomic fluid of the animals, and lysosomal integrity (LI) using the neutral red (NR) retention test. Data showed that TBT and its metabolites (DBT and MBT) bioaccumulated preferentially in pyloric caeca, whereas gonads contained only small quantities. Despite the differences in exposure periods to the contaminated diet and in burdens of butyltins (BTs) ingested by the various contaminated groups, there were no significant differences in body burdens of BTs. Only 6.2+/-2.0% of total ingested BTs were retained in soft tissues of seastars. Even if butyltins were not detected in the coelomic fluid (CF), their detrimental effects have been detected in the phagocytic activity of amoebocytes and their lysosomal retention of neutral red, but no effects were observed on amoebocytes count and their viability. These results show that seastar L. polaris possesses adequate mechanisms to depurate ingested TBT without supporting major disturbances of its immune defence system. By their ability to digest whole contaminated prey and eliminate only dissolved metabolites, L. polaris and other seastars with the same preying mode could play a role of "recycling organisms" in coastal environments where toxicants, such as butyltins and other metallic species are accumulated by bivalves and particularly blue mussels.

Serine proteinase inhibitors from the serpin superfamily have been identified as hemolymph proteins from several groups of arthropods, including horseshoe crabs, crayfish, and insects. In the tobacco hornworm, Manduca sexta, one group of serpins present in plasma is generated by alternate exon splicing from serpin gene-1. We have identified a second serpin gene from this insect, M. sexta serpin-2. A serpin-2 DNA clone was isolated from a fifth instar larval cDNA library. The full-length cDNA is 1.5 kb long and encodes a protein of 381 amino acid residues. Amino acid sequence comparisons with other invertebrate serpins reveal approximately 25-40% identity with serpin-2. An expressed sequence tag from Bombyx mori, which is very similar to M. sexta serpin-2, was identified, and the corresponding full-length cDNA sequence was determined. This silkworm homolog of serpin-2 is 57% identical to M. sexta serpin-2. Recombinant M. sexta serpin-2 was used as an antigen to generate a rabbit polyclonal antiserum. This antiserum recognized a 43 kDa protein present in hemocytes but absent from plasma. Western and Northern blot results revealed that serpin-2 gene expression increased dramatically after larvae were injected with bacteria. In situ hybridization showed that the serpin-2 mRNA is present in granular hemocytes of immune-stimulated larvae. Serpin-2 purified from hemocytes obtained 24 h after injection of larvae with bacteria lacked inhibitory activity for all proteinases tested except for human cathepsin G. The intracellular location of serpin-2 suggests a function for serpin-2 different from the plasma serpin-1 proteins.

Lysozyme is a widely distributed hydrolase which likely plays an important role in bio-defense systems. In the current study, we constructed a baculovirus expression system for the c-type lysozyme cDNA of Japanese flounder (Paralichthys olivaceus) and evaluated the activity of the recombinant protein. This activity was estimated, by turbidimetric assay, to be 7.7U/mg, a value five times higher than that of hen egg white (HEW) c-type lysozyme examined under the same conditions. The optimum pH and temperature for the lytic activity of the Japanese flounder c-type recombinant lysozyme were found to be 5.0-6.5 and 40 degrees C, respectively. Two groupings for fish lysozyme activity are proposed; the first has an optimum pH of approximately 6.0 and the second an optimum pH of above 8.0. Flounder c-type lysozyme was found to possess little lytic activities against Edwardsiella tarda and Streptococcus sp. This latter characteristic may help explain the fact that these two bacterial species are responsible for significant disease problems in cultured Japanese flounder.

Objective: This study is to investigate the functional mimicry by using anti-idiotypic antibodies of enzymes., Methods: Monoclonal anti-idiotypic antibodies against anti-HEL (hen egg-white lysozyme, HEL) antibodies were obtained by fusion of Sp2/0 myeloma cells with spleen cells of syngeneic mice immunized with monoclonal anti-HEL antibodies against HEL's different antigenic epitopes. Then bacteriolysis of the anti-idiotypic antibodies were observed., Results: Eight hybridomas strains secreting anti-idiotypic antibodies were selected and characterized. It was shown that two of eight anti-idiotypic antibodies secreted by two hybridomas (1A10C9 and 2A11C1B3) could mimic HEL catalytic activity to lyse Micrococcus lysodeikticus and that the catalytic effect of mixed anti-idiotypic antibodies of 1A10C9 and 2A11C1B3 was stronger than that of one of them, but less than HEL., Conclusion: The results demonstrated that the anti-idiotypic antibodies that could mimic enzyme activity existed in the idiotype network during anti-enzymatic immune response.

Objective: To elucidate the epitope structure to DNA by identifying antigenic determinants on bacterial DNA bound by anti-DNA antibodies from normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE)., Methods: Sera from NHS and patients with SLE were tested by ELISA for the presence of antibodies to single stranded DNA from calf thymus, Micrococcus lysodeikticus, Staphylococcus epidermidis, Clostridium perfringens, and Klebsiella pneumoniae. To assess binding to conserved and nonconserved determinants, sera were absorbed on DNA-cellulose affinity columns bearing each of the bacterial DNA and then tested for binding to the other DNA antigens., Results: Absorption of SLE sera with any of the bacterial DNA caused a loss of binding to all other bacterial DNA as well as calf thymus DNA. In contrast, absorption of NHS sera with bacterial DNA caused a loss of binding to the DNA on the affinity column with much less effect on binding to the other DNA antigens., Conclusion: These results indicate a marked difference in the specificity of antibodies to bacterial DNA in NHS and patients with SLE. The binding of SLE anti-DNA to predominantly conserved determinants suggests that a shift in patterns of anti-DNA specificity may be associated with the autoimmune state.

Bacterial DNA has potent immunological properties because of its content of immunostimulatory sequences centering on CpG motifs. To investigate whether DNA from various bacterial species differ in these properties, the activity of a panel of DNA was assessed in in vitro cultures of murine spleen cells. This panel varied in base composition and included DNA from Clostridium perfringens (CP), Escherichia coli (EC), Micrococcus lysodeikticus (MC), Staphylococcus aureus (SA), and, as a mammalian DNA control, calf thymus (CT) DNA. In assays of IL-12 and IFN-gamma production as well as B cell mitogenesis, these DNA showed marked differences in their immunostimulatory activity. For both cytokine and B cell responses, EC DNA demonstrated the highest activity while CP DNA had the lowest activity among the bacterial DNA. To determine whether differences in stimulatory capacity resulted from differences in cell uptake, the activity of DNA complexed with lipofectin was tested. While the addition of lipofectin to DNA increased stimulation by all DNA, it did not change the relative potency of the DNA tested. These results indicate that bacterial DNA differ in their immunostimulatory capacity, most likely reflecting their content of CpG motifs. These differences could affect the induction of innate immunity as well as the consequences of infection.

Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds.

Parasitism of lepidopteran host larvae by hymenopteran parasitoids impairs the cellular immune response via expression of polydnavirus genes. Encapsulation of parasitoid eggs is thereby prevented. Parasitized insects are susceptible to opportunistic infections, suggesting that additional components of the immune system are affected. Insects normally respond to infection by inducing the synthesis of an array of antibacterial factors, including cecropins and lysozyme via a NFkappaB/lkappaB-like signal transduction pathway. To characterize the effects of PDVs on the antibacterial immune response, plasma antibacterial activities were assayed in H. virescens larvae infected with the C. sonorensis PDV. Plasma lysozyme activity in Heliothis virescens was reduced in parasitized and PDV-infected larvae after immune challenge. To examine the regulation of lysozyme after CsPDV injection, the Heliothis virescens lysozyme cDNA was cloned. In contrast to plasma lysozyme activity, the 1.1 kb lysozyme mRNA was induced in fat body and haemocytes by known elicitors. The data suggest that CsPDV, like some other viruses, regulates host cell gene expression at the level of translation. We propose that the immunodeficiencies caused by CsPDV injection are caused, in part, by the targeted translational inhibition of specific humoral immune response transcripts.

To elucidate the mechanisms of anti-DNA production, we assessed the binding of sera of normal human subjects (NHS) and patients with SLE to a panel of bacterial and mammalian DNA. Using single-stranded DNA as antigens in an ELISA, NHS showed significant binding to some but not all bacterial DNA, while lacking reactivity to calf thymus DNA. Among bacterial DNA, the highest levels of binding were observed with DNA from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, SLE sera showed high levels of binding to all DNA tested. To evaluate further immunochemical properties of the anti-DNA antibodies, the subclass distribution of these responses was evaluated by subclass-specific reagents. While NHS showed a predominance of IgG2 antibodies to bacterial DNA, SLE sera had a predominance of IgG1 antibodies to these antigens. Together, these results provide further evidence for the antigenicity of bacterial DNA and suggest that NHS and SLE anti-DNA differ in the patterns of epitope recognition as well as mechanisms of induction.

Antibodies to DNA occur commonly in the sera of normal human subjects and bind to nonconserved sites exclusive to DNA from certain bacterial species. These antibodies are primarily IgG2 and differ from anti-DNA antibodies in systemic lupus erythematosus (SLE) sera, which are primarily IgG1 and bind conserved sites on all DNA. To investigate the immune response to bacterial DNA in SLE, antibodies to single-stranded DNA from Micrococcus lysodeikticus (MC) were measured in normal human subjects and in SLE sera by enzyme-linked immunosorbent assay either directly or following absorption by cellulose bearing calf thymus (CT) DNA; isotype-specific reagents were used to assess the IgG2 response. In these assays, SLE sera had a marked deficiency in antibodies specific for MC single-stranded DNA as demonstrated by the reduction of antibody levels by CT DNA cellulose absorption. Furthermore, IgG2 anti-DNA in SLE sera were cross-reactive with MC and CT single-stranded DNA, whereas IgG2 anti-DNA in normal human subjects' sera were specific for the bacterial DNA antigen. Together, these findings indicate that SLE patients have a marked deficiency in the production of antibodies specific for bacterial DNA antigen. This deficiency could predispose to anti-DNA autoantibody production and result from tolerance defects that distort the array of specificities in the B-cell repertoire.

It is proved that in the tick fluid saliva and in the cement plug produced by Rhipicephalus appendiculatus there are bactericidal substances very similar in their activity to the egg lysozyme. The possible role in the transmission and conservation of the specific tick-borne, insensitive to the lysozyme pathogens such as rickettsiae, tick-borne encephalitides virus and borreliae (pathogen of Lyme disease) is discussed. It is supposed, that bactericidal substances in the cement plug protect accumulated in it the tick-borne pathogens (TBE virus, borreliae) from the compete and destructive influence of the vulgar, mainly coccal microflora and from the purulent inflammation, which hinders not only tick feeding but probably specific tick-borne pathogens spreading in the vertebrate host.

Eight species of the genus Micrococcus were studied in details for their antigenic specificities with the aid of the created bank of specific polyclonal antisera to the type strains of Micrococcus Cohn 1872--M. luteus CCM 169, M. varians CCM 884, M. roseus CCM 679 and M. nishinomiyaensis CCM 2140. Immunochemical analysis of 146 strains isolated from various natural and industrial substrata, as well as of 31 collection strains allowed us to reveal antigenic relatedness and distinctions between studied cultures and to accomplish taxonomic distribution of various Micrococcus species. By the immunoduffusion analysis the intraspecific antigenic relationship of M. roseus and M. varians was found, as well as significant antigenic heterogeneity of M. luteus. The results of antigenic analysis of micrococci may be used in the collection work and for the express-diagnostics of these microorganisms.

To assess the human antibody response to bacterial double-stranded (ds) DNA, sera from normal human subjects (NHS) were tested by ELISA for binding to highly purified dsDNA from Micrococcus lysodeikticus (MC). Of 38 NHS tested, 19 demonstrated significant activity to MC dsDNA (defined as an OD380 > 1.0 at a 1:100 dilution of serum) without appreciable binding to dsDNA from Escherichia coli or calf thymus. In competition ELISA, antibodies in NHS to MC dsDNA showed equivalent inhibition by MC ssDNA or dsDNA. Antibody specificity was further evaluated by testing the effects of ionic strength on binding. By ELISA, antibodies in NHS had greater binding to MC dsDNA at high ionic strengths than those in systemic lupus erythematosus sera. These results indicate that NHS have antibodies which bind bacterial dsDNA and extend the range of DNA determinants that can be recognized as foreign by the normal immune system.

Bacterial phagocytosis is a cardinal function of phagocytes. We describe a simple procedure to easily quantify this function using fluoresceinated bacteria. Non-ingested bacteria and those adsorbed to the cell membrane are eliminated by an enzymatic procedure. Only macrophages with ingested fluorescent bacteria are detected, thereby permitting an accurate quantification of the phagocytic process by both spectrofluorimetric measurement and flow cytometric analysis.

Purified cell walls representing a wide variety in teichoic acid and peptidoglycan structure prepared from eight different gram-positive bacterial species induced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 from human monocytes in the presence of 10% plasma or serum. Significant amounts of cytokines began to be produced at concentrations above 100 ng to 1 microgram of cell walls per ml, with maximal production requiring 10 to 100 micrograms of cell wall material per ml. In the absence of plasma, the cytokine-inducing capacity of cell wall preparations was lower by at least an order of magnitude. The serum-derived cofactor was inactivated by heating at 90 degrees C for 30 min, suggesting that the activity is associated with a protein. On the other hand, replacement of normal with hypogammaglobulinemic plasma, inactivation of complement (at 56 degrees C), and blockade by the monoclonal antibody MY4 of the CD14 receptors on monocytes did not inhibit the production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha in the presence of polymyxin B, and macrophages derived from the lipopolysaccharide-insensitive cell line of C3He/HeJ mice also produced this cytokine when stimulated by cell walls. Both peptidoglycan and the soluble glycan-teichoic acid component prepared by an enzymatic method from the same wall preparation exhibited a serum-dependent induction of TNF-alpha from monocytes, while stem peptides and disacharride peptides had only poor, if any, activity. Cell walls may contribute to the septic shock induced by gram-positive bacteria.

It is known that macrophages produce large amounts of eicosanoids during phagocytosis and that pharmacological concentrations of prostaglandin E2 (PGE2) inhibit phagocytosis in several models. However, the physiological effect on phagocytosis of endogenous prostaglandins, produced during CR1- or FcR-mediated bacterial phagocytosis, remains unclear. In this study, we show that indomethacin inhibits the CR1- but not the FcR-dependent phagocytosis of bacteria by rat peritoneal cells in the same range of concentrations that inhibit the synthesis of PGE2, PGI2 and thromboxane A2. An exogenous supply of PGE2 and PGE1 (10(-10) to 10(-8) M) restored the CR1-mediated phagocytosis; higher concentrations were inhibitory. Our data indicate that PGE2 and/or PGI2, produced by rat peritoneal cells, are involved in CR1-dependent bacterial phagocytosis.

Although anti-DNA antibodies are generally considered to be specific markers for systemic lupus erythematosus (SLE), antibodies binding DNA from certain bacterial species can be found in the sera of normal subjects. To characterize the immunochemical properties of these antibodies, the IgG subclass and light chain profile of antibodies to single-stranded micrococcal DNA (MC DNA) in the sera of normal subjects and patients with SLE was determined. The anti-MC DNA response in normal sera was predominantly of the IgG2 subclass with a marked predominance of kappa light chains. In contrast, anti-MC DNA antibodies in SLE sera exhibited all IgG subclasses with a predominance of the IgG1 subclass and both kappa and lambda light chains were represented. These results suggest that antibodies to bacterial DNA in the sera of normal subjects and patients with SLE differ in patterns of immunoglobulin gene expression; the restricted response of normal subjects may be related to the binding to a discrete DNA determinant.

The mechanisms of phagocytized materials (synthetic HEMA particles, micrococci) by earthworm coelomocytes were examined. Despite the fact that coelomocytes have a high phagocytic activity, the chemiluminescent assay did not reveal increased production of oxygen radicals. Ultrastructural changes of engulfed particles observed by electron microscopy may be caused by intracellular lytic enzymes.

The purpose of this study was to compare the phagocytosis of Aeromonas hydrophila, Edwardsiella ictaluri, Edwardsiella tarda and Micrococcus luteus by channel catfish neutrophils. Various aspects of opsonization effect and bactericidal ability of channel catfish neutrophils were investigated. Percent phagocytosis ranged from a low of 1% to a high of 91%. The highest percent phagocytosis and phagocytic indices routinely occurred with normal serum and were highest for M. luteus (91.78%, 55.25) and A. hydrophila (87.52%, 43.60). In all cases, the percent phagocytosis and phagocytic index was lowest in assays without serum. Channel catfish neutrophils displayed a bactericidal/static ability for each bacterium tested except E. tarda. Neutrophils exhibited a greater inhibitory capacity for A. hydrophila and M. luteus than for the other bacteria when in the presence of normal or inactivated catfish serum.

Auranofin (AF), D-penicillamine (D-pen) and thiola are prescribed as disease-modifying drugs in the treatment of rheumatoid arthritis (RA). We have shown here that auranofin, 10(-8) to 10(-6) M, D-penicillamine, 10(-6) to 10(-3) M, thiola, 10(-7) to 10(-3) M, and the tripeptide thiol, glutathione, 10(-6) to 10(-3) M, enhanced f-met-leu-phe-induced lysosomal enzyme release and the phagocytic uptake of bacteria by up to 40%. The previously reported inhibitory effects of AF were only observed at concentrations in excess of those likely to be available to effector cells in vivo. The stimulatory effects of thiola and D-pen occurred at concentrations likely to be available to effector cells in vivo and, therefore, may be of greater clinical relevance. There is evidence that the drugs used in this study exert their effects via a thiol moiety and their therapeutic effect is preceded by an elevation of intracellular thiol levels.

Morphological mutants of Micrococcus lysodeikticus (luteus) were isolated by treatment with N-methyl-N'-nitro-N-nitrosoguanidine. They occurred on plates in large, regular cell packets, whereas the parent cells usually grew as groups of two or four cells or as short chains. The mutants required a much higher concentration of Mg2+ for growth than the parent cells. The concentrations of Mg2+ and other components of the culture medium tested did not significantly affect the morphology of either the parent or mutant strains. The mutant strains were not agglutinated by antiserum to M. lysodeikticus, which mainly interacts with teichuronic acid on the cell surface, and chemical analysis of isolated cell walls of the mutants indicated the absence of teichuronic aicd. No significant differences were detected between the parent and mutant strains in the amounts of other cell wall components, e.g., peptidoglycan, protein, and teichoic acid. They possible roles of teichuronic acid in cell separation and attachment of divalent cations are discussed.