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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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.

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.

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.

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.

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.

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.

Lysates obtained from amoebocytes of Limulus polyphemus, the horseshoe crab, showed gel formation after the addition of bacterial endotoxin. In contrast to living gram-negative bacteria, viable gram-positive microorganisms did not cause gelation of lysate. Nevertheless, peptidoglycan isolated from the cell walls of various gram-positive organisms did induce the reaction. However, the activity of peptidoglycan was 1,000 to 400,000 times less than that of Escherichia coli lipopolysaccharide. After exposure to lysozyme, peptidoglycan no longer gelled amoebocyte lysate, therefore apparently excluding endotoxin contamination. Gelation of amoebocyte lysate by endotoxin or peptidoglycan was inhibited by different concentrations of sodium polystyrolsulfonate. Whereas these studies confirm the specificity of the Limulus test for bacterial endotoxins, they also indicate that other substances of bacterial origin should be investigated for their ability to gel amoebocyte lysate.

The bacteriolytic and bactericidal effects of the human proteinases cathepsin B, cathepsin D, cathepsin G, and elastase were investigated. Cathepsin G and elastase were 5 to 10% as active as egg white lysozyme in the lysis of Micrococcus lysodeikticus. All four enzymes slowly lysed the lysozyme-resistant Staphylococcus aureus. The gram-negative Acinetobacter 199A was rendered sensitive to lysozyme by all of the proteinases. Only elastase caused marked proteolysis of the outer membrane, which would permit access by lysozyme to the underlying peptidoglycan. When the surface layer of regularly arranged a protein was removed, however, the outer membrane proteins became susceptible to the other proteinases. Cathepsin G, elastase, and cathepsin D were bactericidal to Acinetobacter 199A. The bactericidal activity of cathepsin D was shown to be dependent on enzymatic activity, unlike that of cathepsin G, which was related to its cationic nature.

The injection of thioglycollate medium into the peritoneal cavity of the mouse induces high levels of macrophage fibrinolytic activity due to the production and secretion of a plasminogen activator, a trypsinlike serine protease, which is absent in unstimulated macrophages. Intraperitoneal injection of endotoxin or mineral oil can stimulate only a fraction (<10%) of the fibrinolytic activity of thioglycollate cells, similar to the partial stimulation (<10%) seen 1-2 days after phagocytosis of latex or SRBC by unstimulated macrophages. The endotoxin-stimulated macrophages contain and release relatively low levels of plasminogen activator, but these primed cells can be triggered to produce and secrete high levels of enzyme, by phagocytosis of latex. Under conditions where the plasminogen activator is induced and secreted, there are no effects on the production and/or release of lysozyme or intracellular acid hydrolases, Discovery of a two-stage procedure for inducing macrophage plasminogen activator made it possible to study the role of cell priming and phagocytosis separately. Endotoxin was a more effective priming agent, weight for weight, than lipid A:BSA complex. Secretion of the plasminogen activator was induced only by thioglycollate, or endotoxin and latex. In situ fibrinolysis was induced by these agents and mineral oil, BCG, and fetal calf serum, in decreasing order of effectiveness. Phagocytosis of latex in all cases except thioglycollate stimulation, increased fibrinolytic activity from three- to sixfold. Latex and a variety of other particles such as M. lysodeikticus, aggregated gamma-globulin and immune complexes showed dose-dependent stimulation of fibrinolysis by endotoxin-primed macrophages. Although the initial phagocytic trigger was not specific for the substance employed, the ability to induce a sustained response depended on the persistence of the phagocytized particle within the cell. Fibrinolysis and secretion of plasminogen activator continued at high levels for at least 9 days after uptake of latex, a nondigestible particle, whereas plasminogen activator was secreted only transiently after ingestion of rapidly digested M. lysodeikticus. The induction of plasminogen activator secretion provides a mechanism by which the activated macrophage can exert a selective effect on its extracellular environment.

Triton X-100-insoluble residues from Micrococcus lysodeikticus membranes were analyzed by crossed immunoelectrophoresis after dispersal of the residues in sodium dodecyl sulfate (SDS). Conditions which produce no obvious distortion of the immunoprecipitate profile and which allow qualitative and quantitative analyses of the antigens present in the extracts are described. Two main antigens were detected; these were identified as succinate dehydrogenase (EC 1.3.99.1) and adenosine triphosphatase (EC 3.6.1.3). As determined by peak area estimations, the maximal release of succinate dehydrogenase and of adenosine triphosphatase from Triton X-100-insoluble membrane residues occurred at protein/SDS ratios of about 4.3:1 (0.2% SDS) and 6.8:1 (0.13% SDS), respectively. A comparison of enzyme activities of SDS extracts with those of untreated, control Triton X-100-insoluble membrane residues indicated that both the succinate dehydrogenase and the adenosine triphosphatase antigens were released with a full (or enhanced) catalytic potential at or below concentrations of SDS required to effect maximal solubilization of the enzyme in question. Evidence is also presented to suggest that the more acidic of the two components detected by crossed immunoelectrophoresis for the heterogeneous adenosine triphosphatase antigen is more sensitive to SDS than is the other. Both succinate dehydrogenase and adenosine triphosphatase lost catalytic activity and were denatured at protein/SDS ratios lower than 3.4:1.

The antibody response of a single outbred rabbit was studied throughout three rounds of injections with Micrococcus lysodeikticus vaccine over a 31-mo period. The first-round response was characterized by a vigorous anti- micrococcus response and a strong anti-IgG rheumatoid factor response. The second-round response consisted of a triad of interacting molecules: anti- micrococcal antibodies, autoanti-idiotypic antibodies specific for distinct clonotypes of the first-round anti-micrococcal antibodies, and Fc-specific anti-IgG rheumatoid factor. The interacting triple complex was detected because of the formation of an immune complex that became insoluble upon dilution of the serum. Complex formation was inhibited in the presence of saccharide compounds known to be major immunodominant determinants of the micrococcal cell-wall carbohydrate polymer. The same saccharides did not affect the reaction of rheumatoid factor with IgG. Direct-binding radioimmunoassays ruled out mediation of the dilution-precipitation reaction by soluble micrococcal antigens. Specific absorption of rheumatoid factor inhibited the dilution-precipitation reaction. Auto-anti-idiotypic antibodies were specifically purified from second-round sera, directly confirming the presence of these antibodies. Suppressive effects of auto-anti-idiotypic antibodies on distinct antibody clonotypes were shown by gel isoelectric focusing of first-, second-, and third-round sera. Clonotypes expressed in the first round of immunizations were reduced in quantity or absent when auto-anti-idiotypic antibodies were detectable. Greatly enhanced levels or initial synthesis of new clonotypes of anti-micrococcal antibodies were detected during the period of auto-anti-idiotype synthesis. The third-round sera, devoid of detectable auto-anti-idiotype, contained clonotypes characteristic of both first- and second-round antisera. Thus, auto-anti- idiotypic-mediated suppression appeared to be reversible. The data are interpreted as lending strong support for concepts of autoregulation of immune processes in normal outbred animals via an idiotypic network.

Sera from patients with verified systemic staphylococcal infection contained antibodies reactive with peptidoglycan (PG) from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Micrococcus lysodeikticus, Bacillus subtilis, and Escherichia coli. The presence of anti-PG cross-reactive antibodies was verified in patient sera by inhibition studies with the various bacterial PGs. Antibodies to nonstaphylococcal PGs were also elevated in sera from rabbits immunized with S. aureus PG. Antibodies to S. aureus PG were removed with the synthetic peptide analogs of S. aureus PG, the L-Lys-D-Ala-D-Ala, L-Lys-D-Ala, and (Gly)5 determinants, as well as with an S. aureus PG peptide fragment containing the determinants D-Ala-D-Ala and L-Lys-D-Ala. Isolated antibodies to the PG peptides, both synthetic and native, were reactive with S. aureus and S. epidermidis PGs. The antibodies to the D-Ala-D-Ala and the L-Lys-D-Ala determinants were also reactive with S. pyogenes PG, but not with PGs from M. lysodeikticus, B. subtilis, and E. coli.

Lysozyme release from alveolar macrophages is stimulated by exposure to particles, such as latex and zymosan, and to bacteria. Rat alveolar marcophages contain 10-fold-greater intracellualr concentrations of lysozyme and release more lysozyme after stimulation than rat blood neutrophils. During 30 min of incubation in vitro, alveolar macrophages kill more than 99% of Micrococcus lysodeikticus in the incubation mixture, whereas neutrophils kill approximately 50% of the bacteria. The bactericidal capacity of alveolar macrophages for M. lysodeikticus exceeds that of neutrophils at all bacteria-to-cell ratios tested. This bacterial killing by alveolar macrophages is inhibited when specific rabbit antirat lysozyme serum is added to the incubation mixture. Electron microscopy studies indicate that bacterial killing occurs extracellularly. Initial degradation of bacteria occurs within 5 min, and lysis is complete by 25 to 30 min. Phagocytosis of lysed bacteria is maximum after 25 to 30 min. The greater quantities of lysozyme, both intracellularly and released into the extracellular environment by alveolar macrophages, suggest that this factor may be a mechanism by which alveolar macrophages contribute to pulmonary defense.

The experiments aimed at evaluating the optimal parameters in the chemo-immunotherapeutic treatment of the L1210 lymphoid leukaemia grafted to [female BALB/c (H2d) X male DBA/2 (H2d)]F1 hybrid mice, hereafter referred to as CDF1 mice. In vitro irradiation of leukaemic ascites cells by X- or gamma-rays and subsequent inoculation in mice showed that optimum immunogenicity is radiation dose-dependent. Grafting mice with 10(7) leukaemic ascites cells irradiated at optimum dose (80 GyX- or gamma-rays) delays mortality of the animals when challenged later with untreated L1210 cells, but is unable to cure mice. By contrast, specific immunoprophylaxis induced by Micrococcus, complement-triggering polysaccharides or BCG and irradiated leukaemic cells was able to protect mice against grafts of 10(4) L1210 cells. The i.p. route was notably superior to the i.v. route. When mice bearing advanced L1210 tumour were treated by chemotherapy (12 mg/kg of BCNU) on Day 6.5 after grafting 10(4) L1210 cells and subsequently treated by immunotherapy, a very high percentage (up to 90%) of mice with 10(8) leukaemic cells could be cured by repeated 1mg injections of bacterium or polysaccharide, and challenge with irradiated leukaemic cells was unnecessary. Because of the high cure rate obtained, the very regular response pattern and the non-pathogenicity, the bacterium Micrococcus lysodeikticus would seem a promising new candidate for chemo-immunotherapeutic antitumour strategies.

Anti-idiotypic antibodies (Ab2) were raised in allotype-matched rabbits against anti-carbohydrate or anti-tobacco mosaic virus antibodies (Ab1). Several Ab2 were purified and injected into a third series of rabbits III which synthesized antiantiidiotypic antibodies (Ab3). Antigen was then given for the first time in those rabbits who had synthesized Ab3. The specific antibody synthesized in rabbits III was called Ab1'. Anti-idiotypic antibodies were raised against purified Ab3 antibodies (Ab4). In most cases, Ab1' antibodies are sharing idiotypic specificities with Ab1. Ab3 did not react with antigen but shared idiotopes with Ab1 and Ab1' because Ab4 antibodies, which are anti-idiotypes to Ab3 do recognize specifically Ab1 and Ab1' antibodies belonging to the same chain of immunization. It seems therefore that Ab3 looks idiotypically like Ab1 and Ab4 displays the same behaviour as Ab2. A general view of the functioning of the immune system is presented.

Rabbits capable of producing antibodies of restricted heterogeneity in response to Micrococcus lysodeikticus are equally capable of producing antibodies of restricted heterogeneity to bovine serum albumin. These antibodies are produced when animals are simultaneously injected with micrococcus and BSA and their specificity is restricted to a small number of epitopes. These results suggest that micrococcal vaccines can induce the restriction of heterogeneity in antibodies raised against totally unrelated antigens.

Naturally induced auto-anti-idiotypic (AAI) antibody responses specific for antimicrococcal antibody idiotypes were detected in 42% of the rabbits in a family immunized with Micrococcus lysodeikticus. The natural AAI response of each rabbit recognized only a portion (11-41%) of that individual's total antimicrococcal antibody population. Cross-reactions of idiotypes were observed within the group of rabbits exhibiting natural AAI responses. Examination of the basis for the cross-reactions showed that the natural AAI antisera recognized identical idiotopes on the antimicrococcal F(ab')2 fragments from each rabbit that made an AAI response. The cross-reactive idiotopes were shown to be of paternal origin and were found in the antimicrococcal antibodies of each offspring. The data strongly support the idiotypic network concept that naturally induced AAI responses may occur routinely in outbred normal individuals as a result of antigenic stimulation. Further, the data suggest that the induction of regulatory AAI antibody responses in outbred rabbits may depend on the expression of particular germ line idiotopes.

By crossed immunoelectrophoresis with membrane antiserum, 17 antigens have been detected in fractions from plasma membranes of M. lysodeikticus solubilized with Triton X-100. Absorption tests with protoplasts have demonstrated that eight of the antigens are expressed on the surface. Of these antigens the major one has been identified as a succinylated mannan. Five of the principal immunoprecipitates unaffected by absorption with protoplasts were shown by zymograms to possess the following enzymic activites: succinate dehydrogenase (EC 1.3.99.1), ATPase (EC 3.6.1.3), NADH dehyrogenase (EC 1.6.99.3)(two separate components), and malate dehydrogenase (EC 1.1.1.37). These enzymes or enzyme-complexes are, therefore, not expressed on the outer surface of the protoplast membrane.

Specific idiotypie can be induced in randomly chosen rabbits by preimmunization with anti-idiotypic antibodies (Ab2). Rabbits that synthesize anti-anti-idiotypic antibodies (Ab3) when injected with antigen produce antibodies that display idiotypic specificities that are also found on the starting idiotype. When female rabbits actively producing Ab3 are crossed with naive males, a significant proportion of the offsprings (approximately 40%) produce antibodies that were idiotypically cross-reactive with the starting idiotype, as compared to 3% of the controls. This conclusion was obtained using 5 female rabbits and their 32 surviving offspring. Maternal idiotypes have therefore strong immunoregulatory properties and influence the emergence of the available idiotypic repertoire.

A simple test for the serological separation of staphylococci from micrococci is described, which is based on the quite different cell wall peptidoglycan structures of these two genera. Antisera to (pentaglycyl-epsilon-amino-n-hexanoic acid)20-albumin agglutinated without exception all staphylococci and gave no positive reaction with micrococci or other bacterial cells. To obtain a good reaction, it was necessary to extract the cells with hot trichloroacetic acid for 30 min. Antisera to (tri-L-alanyl-epsilon-amino-n-hexanoic acid)22-albumin reacted strongly with micrococci containing oligo-L-alanine bridges in their peptidoglycan, but did not agglutinate staphylococci or other bacteria lacking alanine interpeptide bridges.

Anticarbohydrate antibodies (Ab1) were isolated from a rabbit hyperimmunized with Micrococcus lysodeikticus and injected into allotype-matched rabbits in order to obtain specific anti-iodiotypic antibodies (Ab2). Ab2 was isolated by means of a Sepharose column coupled to the anticarbohydrate antibodies and was injected into two allotype-matched rabbits. These latter rabbits produced specific anti-anti-idiotypic antibodies (Ab3) probably sharing idiotypic specificities with Ab1. However, these Ab3 did not react with the antigenic carbohydrate moiety of bacteria. The two rabbits that had produced Ab3 were then immunized with M. lysodeikticus and synthesized anticarbohydrate antibodies (Ab1') bearing idiotypic specificities similar to those of Ab1. The immune repertoire which is effectively expressed in one individual depends not only on the antigenic stimulation but also on the previous idiotypic history of the individual. These data support the concept that the immune system is a functional idiotypic network.

Micrococcus luteus IFO 3333 cells, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. The surface of a M. luteus cell consisted of two or three areas with borders--the rough and the smooth areas, or the rough, the slightly rough, and the smooth areas; fluffy materials were clearly seen in the rough area. Gold particles were observed uniformly and densely on the whole cell surface. However, either mild acid treatment or mild Smith degradation of the cells altered the fluffy rough area to a rough one, and extremely decreased the agglutinability and the binding of protein A-gold particles. Teichuronic acids appeared to be distributed uniformly on the whole cell surface of M. luteus IFO 3333.

Fresh, defibrinated human blood (80 vol%, i.e., 80% [vol/vol] of a 2-ml final assay volume) from two healthy adult donors killed "delayed serum-sensitive" (DSS) and "promptly serum-sensitive" (PSS) strains of Serratia marcescens, PSS control strain Escherichia coli C, Bacillus subtilis strain ATCC 6633, and Micrococcus lysodeikticus ATCC 4698 in a kinetic manner comparable to that of fresh human serum (80 vol%). However, heat-inactivated (56 degrees C, 30 min), defibrinated human blood revealed markedly reduced or a total lack of beta-lysin activity against the B. subtilis assay strain. Similarly, lysozyme activity of defibrinated blood was diminished somewhat by heat treatment, as determined with the M. lysodeikticus assay strain. Addition of 500 mug of sodium polyanetholsulfonate (SPS) per ml to 80 vol% of fresh, defibrinated human blood completely neutralized blood bactericidal activity against all assay strains of S. marcescens, E. coli C, and B. subtilis; however, SPS at this concentration failed to abolish lysozyme activity for prolonged periods of incubation. Addition of 500 mug of sodium amylosulfate (SAS) per ml to 80 vol% of fresh defibtinated human blood resulted in protection of cell inocula of DSS strains of S. marcescens only; SAS failed to protect cell inocula of the PSS strains of S. marcescens, E. coli C, B. subtilis, and M. lysodeikticus for extended periods of observation. Based on these data, it is recommended that blood culture specimens that are first drawn into specimen containers (such as Vacutainer tubes or the like) at the patient's bedside, and which contain >/=250 mug of SPS per ml, be diluted into suitable broth media with at least >/=250 mug of SPS per ml by the receiving laboratory within 2 to 4 h after procurement of the specimen. This procedure would ensure continued, adequate neutralization of the specimen's inherent beta-lysin, lysozyme, and complement- and antibody-mediated bactericidal activities.

Binding of lectins to microbial cell walls was investigated by fluorimetric titration and Scatchard plot. Data were correlated with agglutinability. Concanavalin A and lectins of wheat germ, soybean, pea, lentil, and peanut were tested against Escherichia coli. Micrococcus luteus, Lactobacillus plantarum, and Bacillus subtilis. In cases where binding occurred, it was either nonspecific or positively cooperative. Agglutination was observed only in those combinations of lectin and microorganism that showed positive cooperative binding, suggesting a definite relation between binding and agglutination. Lectins binding to the same carbohydrate did not necessarily bind to the same microorganism, confirming the complexity of the lectin-receptor bond.

In order to test the relative immune competence of a minor VH subgroup (y), the concentrations of rabbit Ig bearing the VH subgroups a and y were determined in experimentally immunized rabbits. Three groups of three rabbits each were immunized with either Micrococcus luteus, bovine serum albumin or an alloantigenic determinant (b4) of the rabbit kappa light chain. The ratios of VHy to VHa Ig in the pre-immune sera ranged from 1:8 to 1:283. In five of the nine rabbits, the VHy to VHa ratios in affinity purified antibody (Ab) closely reflected the ratios of VHy to VHa in pre-immune serum. Of the remaining four rabbits, three produced disproportionately low amounts of VHy Ab, when compared to pre-immune values, while one rabbit produced virtually no VHy Ab. Regardless of the levels of VHy and VHa Ab detected, the kinetics of Ab production by each subgroup during the course of the immune response were similar to each other. When total serum levels of the subgroups were analysed, all rabbits displayed increases (6-450%) in total serum levels of both VHa Ig and total IgG following immunization, while in eight out of nine rabbits the level of VHy Ig remained unchanged. Our observations suggest that the immune repertoire of the minor VHy subgroup is sufficiently diverse to generate VH regions which react with a variety of antigens, and in five out of nine rabbits, VHy Ab-producing clones compete well with VHa Ab-producing clones. In addition, it appears that clones of the major and minor VH subgroups, once triggered, are under similar regulatory controls with respect to specific Ab, but differ with respect to non-specific Ig production.

Pupae of the silk moth, Samia cynthia, were found to contain an inducible antibacterial activity in their hemolymph. This immunity response was provoked by primary infections with either Escherichia coli K-12 or Enterobacter cloacae. In both cases the antibacterial activity was directed chiefly towards E. coli. During standard conditions, 1% of hemolymph could kill 10(3) to 10(4) viable E. coli, strain D31, within 5 min. A lower level of antibacterial activity was induced by injections of a sterile salt solution. The killing of strain D31 followed single-hit kinetics, and increasing rate constants were obtained for increasing amounts of hemolymph. The reaction was sensitive to pretreatment with trypsin and it was protected by reducing agents. The activity was inhibited by microgram quantities of lipopolysaccharide (LPS) prepared from certain LPS mutants of E. coli K-12. A comparison of the susceptibility showed that "heptose-less" LPS mutants were more sensitive to killing than other strains. During standard conditions hemolymph will lyse both E. coli and Micrococcus lysodeikticus. Lysis of E. coli followed a multi-hit kinetics and it was inhibited by LPS, whereas lysis of M. lysodeikticus was unaffected by LPS. Hemolymph was fractionated on Sephadex G-200, and the lytic activities were recovered in partly overlapping peaks. Reconstitution with pooled fractions gave synergistic effects with the killing assay.

Anti-phosphatidylglycerol and anti-cardiolipin antisera were prepared in rabbits by using phospholipids purified from Micrococcus lysodeikticus. Anti-phosphatidylglycerol antibodies were found in antisera when either phosphatidylglycerol or cardiolipin were used as immunogens, but adsorption studies indicated they were not similar. Antibodies which reacted with phosphatidylinositol and phosphatidic acid were also found in the anti-cardiolipin antiserum. Structures of the antigenic groups in phosphatidylglycerol and cardiolipin are suggested from cross-reaction and adsorption studies. Adsorption studies with pure phospholipid antigens indicated the importance of the spacial orientation of phospholipid haptens for immunological reactivity.

A comparative study of the effects of BCG, Micrococcus lysodeikticus, and a series of structurally related polysaccharides (complement triggers) on the non-specific and specific immune resistance against L1210 lymphoid leukaemia was carried out and commented on. In contrast with authors of earlier reports, we were unable to generate any effective non-specific or specific immunotherapy after the graft of 10(4) leukaemic cells to 8--10-week-old CDF1 mice. However, when mice were prevaccinated with irradiated (8 krad X-rays) cultured cells combined with 1 mg of bacterium or polysaccharide one month before grafting 10(4) cells, they were given an immunoprotection that was more pronounced with the i.p. than with the i.v. route. Prevaccinated mice were afforded a stronger immunoprotection when boosted repeatedly with 1mg injections of bacterium or polysaccharide after tumour challenge.

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Conventional rabbit antiidiotypic antibodies were raised against a private rabbit anti-tobacco mosaic virus (TMV) idiotype. These rabbit antiidiotypic antibodies were covalently coupled to lipopolysaccharide and then injected into BALB/c mice. As compared with controls, these mice, which have never seen the antigen, synthesized anti-TMV antibodies that are strongly idiotypically cross-reactive with the starting rabbit idiotype. Monoclonal anti-TMV antibodies were prepared from these mice. Furthermore, xenogeneic or syngeneic antiidiotypic antibodies, raised against these monoclonal anti-TMV antibodies, recognized specifically the rabbit idiotype. Rabbit antiidiotypic antibodies alone can induce the same effects, but the concentration of anti-TMV antibodies is lower.

Normal fresh and heat-inactivated (56 degrees C, 30 min) human sera (80 vol%, i.e., 80% [vol/vol] of a 2-ml assay volume) killed Bacillus subtilis ATCC 6633 cell inocula of 1.5 x 10(4) colony-forming units per ml within 1 to 2 h after exposure. The B. subtilis assay strain proved slightly and reversibly susceptible to 5 mug of egg white lysozyme per ml. Seitz filtration of fresh human serum completely removed beta-lysin activity; significant amounts of serum lysozyme were removed as well, as determined with the bioassay strain Micrococcus lysodeikticus ATCC 4698. However, bactericidal activity of human serum via classical or alternative complement pathway activation remained intact. Addition of 0.01 M dithiothreitol to fresh human serum abolished beta-lysin activity, but not that of serum lysozyme. Chelation of fresh and heat-inactivated human serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid, but not with 0.01 M ethylenediaminetetraacetic acid, markedly retarded beta-lysin activity; however, lysozyme activity remained unaffected. Chelation of serum with 0.01 M MgCl(2) + 0.01 M ethylene glycol tetraacetic acid + 0.01 M CaCl(2) completely abrogated beta-lysin activity, but not that of lysozyme. Absorption of human serum with 10 mg of bentonite per ml (10 min, 37 degrees C) completely removed beta-lysin and lysozyme activity, but failed to affect serum bactericidal activity against Escherichia coli control strain C. Reconstitution of 50 vol% of bentonite-absorbed serum with 40 vol% of heat-inactivated human serum restored both beta-lysin and lysozyme activity. Addition of either 63 to 500 mug of sodium polyanetholsulfonate per ml or 63 to 500 mug of sodium amylosulfate per ml to 80 vol% of fresh human serum completely neutralized beta-lysin activity for the entire observation period of 22 h.

The naturally occurring (nonspecific) proteinase inhibitor which occurs in the alpha globulin fraction of immune rabbit serum was separated from specific staphylococcal and micrococcal antiproteinases by treatment of the antiserum with 2-ethoxy-6-9-diaminacridine. The 2-ethoxy-6-9-diaminacridine-treated serum could then be used to classify these bacterial proteinases by testing directly on casein agar without prior electrophoresis of the serum.