Innate immune response of channel catfish Ictalurus punctatus mannose-binding lectin to channel catfish virus (CCV).

The channel catfish virus (CCV) is a pathogenic herpesvirus that infects channel catfish Ictalurus punctatus in pond aquaculture in the southeastern USA. Mannose-binding lectin (MBL), an innate immune protein, could play an important role in the innate response of channel catfish by binding to CCV. Cell cultures of CCV were grown in channel catfish ovary cells (CCOC). A dot-immunoblot enzyme-linked immunosorbent assay was done to determine the binding ability of 5 mo old channel catfish serum MBL (26.2 µg ml-1) to CCOC infected with CCV. Two separate nitrocellulose membrane blotting techniques were done using uninfected and infected CCOC. The uninfected CCOC decreased by 29.3 and 33.4% in their binding of channel catfish MBL when compared with infected CCOC using the 2 membrane procedures. The combined average binding ability of channel catfish MBL towards infected CCOC was therefore 31.4% greater when comparing the infected and uninfected CCOC. Normalization equation values of MBL for the 5 mo old catfish were compared for the 2 membrane binding procedures. The 2 normalization values were very close (142 and 150) in binding ability of MBL to the infected CCOC. The 5 mo catfish serum had twice the concentration of MBL (26.2 µg ml-1) compared to 7 mo catfish serum (13.2 µg ml-1), and the binding percentage of 5 mo serum was 2.4 times greater in infected than in uninfected cells. This demonstrates that the binding of channel catfish serum MBL to CCV is concentration dependent and is related to serum concentrations of MBL.

Spleen index and mannose-binding lectin levels in four channel catfish families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri.

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.

Antitumor cell activity in vitro by myristoylated-peptide.

New anticancer drugs are needed to support cancer treatment. Cancer involves the uncontrolled proliferation of tumor cells, and many anticancer drugs are agents that inhibit tumor cell growth. The antitumor cell N-myristoylated-peptide is a natural product purified from Heliothis virescens insect hemolymph with essentially no cytotoxicity against human foreskin fibroblast cells. A synthesized structure of six amino acids (Myristoyl-Cys-Ala-Val-Ala-Tyr-[3 methyl]His-OMe; M.W. 902.2 Da) was screened at 10 µM for in vitro antitumor activity against 56 human tumor cell lines by the National Cancer Institute (NCI). The NCI found that 34 of the 56 tumor cell lines (representing eight major tumor types) were sensitive (> 50% growth inhibition of tumor cells) to the myristoylated-peptide. The best overall antitumor activities (>60% average growth inhibition) were seen against 17 tumor cell lines for non-small cell lung cancer, leukemia and melanoma and for eight other tumor cell lines. The NCI finds that their in vitro screen is an effective selector of compounds with in vivo antitumor activity.

Susceptibility in vitro of Epstein-Barr Virus to myristoylated-peptide.

The anti-Epstein-Barr Virus (EBV) myristoylated-peptide (M.W. 916.2Da) is a natural product isolated from Heliothis virescens insect larval hemolymph (blood) that essentially has no cytotoxicity against human foreskin fibroblast cells. A (3 methyl only) version (M.W. 902.2 Da) of the structure was synthesized and tested for in vitro anti-EBV activity and cytotoxicity. The N-terminal end is lipophilic and used to get the compound across the cell membrane. The C-terminal end with its ring-shaped structures is likely used to inhibit DNA synthesis. The synthetic compound inhibited DNA synthesis/replication of EBV in Akata cells (B-lymphocyte from Burkitt's lymphoma patient) in in vitro tissue culture. A DNA hybridization assay for anti-EBV activity using the Akata B-cell and two cytotoxicity assays using human foreskin fibroblast cells were done with the synthetic peptide. Effective concentration (EC90) at 20 microM inhibited viral replication by 90%. The EBV, known as Human Herpesvirus-4 (HHV-4) of the Herpesviridae family, has been described as a cancer-promoting double-stranded DNA virus that may also be involved in autoimmune disease. There are no antiviral drugs in clinical use for diseases caused by the EBV.

Isolation of lysozyme and an antifungal peptide from sea lamprey (Petromyzon marinus) plasma.

The sea lamprey (Petromyzon marinus) belongs to the most primitive class of fish and has only innate immunity. The innate immune factors, lysozyme and an antifungal peptide, were isolated from sea lamprey plasma. Sea lamprey plasma (40.1mg protein/ml) was assayed for lysozyme activity by gel diffusion assay. Using hen egg white lysozyme standards, plasma concentration of lamprey lysozyme was 5microg lysozyme/mg total protein. The presence of lysozyme in such high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. Lysozyme and the antifungal peptide were isolated by low molecular weight gel filtration chromatography from sea lamprey plasma. Gel filtration chromatography yielded two peak tubes containing lysozyme (1microg/211microg total protein) and antifungal peptide (1microg/66microg total protein). Lysozyme and antifungal activity of each fraction were determined by well diffusion assay using Gram-negative bacteria, Gram-positive bacteria and two fungal species. The molecular weight of lamprey lysozyme was 14.3kDa. The sea lamprey lysozyme was effective against Gram-positive bacteria but not against Gram-negative bacteria or fungi. Molecular weight of the antifungal peptide was approximately 3000Da. Antifungal plasma activity was seen against Penicillium notatum and Aspergillus flavus. No plasma antibacterial peptide was found.

Isolation of mannose-binding C-type lectin from sea lamprey (Petromyzon marinus) plasma and binding to Aeromonas salmonicida.

The sea lamprey (Petromyzon marinus) is a parasitic cartilaginous fish of the North American Great Lakes and a predator of many bony fish species of commercial importance to the fishing industry. Mannose-binding C-type lectin (MBL) was isolated by mannan-agarose affinity chromatography from sea lamprey plasma. Mannose-binding lectin has not before been identified and quantitated in the plasma of this sea lamprey species. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35kDa and 65kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. Amino acid composition analysis (mol%) of the purified lamprey MBL found high amounts of histidine, threonine, tyrosine and phenylalanine present when compared with three other vertebrate MBLs. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261mug of MBL/ml of plasma. Plasma protein concentration was 40.1mg/ml. Lamprey MBL was present then in plasma at 6.5mug MBL/mg total protein. The sea lamprey MBL also specifically binds to mannose on the surface of the pathogen Aeromonas salmonicida. The presence of MBL in high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. This study describes the presence of MBL in sea lamprey plasma and evidence for a C-type lectin complement pathway of innate immunity.

Comparative study of mannose-binding C-type lectin isolated from channel catfish (Ictalurus punctatus) and blue catfish (Ictalurus furcatus).

Mannose-binding C-type lectin (MBL) was isolated from channel catfish (Ictalurus punctatus) NWAC 102 and 103 strains, blue catfish (Ictalurus furcatus) D+B and Rio Grande strains, hybrid catfish (channel catfish female NWAC 103 x blue catfish male D+B) sera, and purified by affinity chromatography from channel catfish Norris strain serum. Reduction of purified channel catfish MBL with 2-ME yielded a single band of 62 kDa by SDS-PAGE and Western blot analysis using guinea pig anti-MBL IgG as primary antibody. Channel catfish NWAC 102 strain, channel catfish NWAC 103 strain and hybrid catfish sera had molecular masses of 63 kDa for MBL. Blue catfish (D+B strain) serum MBL had a molecular mass of 66 kDa. Rio Grande blue catfish serum MBL had a molecular mass of 65 kDa. Amino acid composition analysis (mol%) of the affinity-purified channel catfish MBL found a high content of serine present. Functional binding studies of channel catfish and blue catfish MBLs binding to Edwardsiella ictaluri were done using a dot-immunoblot ELISA method. A dot-immunoblot ELISA binding assay was done to compare nine different strains and species of channel catfish and blue catfish for their levels of serum MBL. Blue catfish had higher levels of MBL than did the various strains of channel catfish tested. MBL could be used as a genetic marker for selection of disease resistance in the different strains of catfish used in aquaculture. This study describes the presence of serum MBL in catfish and evidence for a C-type lectin complement pathway of innate immunity.

Isolation of mannose-binding C-type lectin from Heliothis virescens pupae.

A mannose-binding C-type lectin (MBL) was isolated by affinity chromatography from Heliothis virescens immune pupal hemolymph. The immune pupal hemolymph was obtained after bacterial injection of live Enterobacter cloacae bacteria. MBL in mammals acts as an opsonin for phagocytosis and activates the lectin complement pathway of the innate immune response, which leads to killing of gram-negative bacteria and enveloped viruses. The affinity-purified and reduced pupal MBL showed a single band of 36 kDa by SDS-PAGE (12% gel). A dot-immunoblot ELISA (using guinea pig anti-MBL IgG as primary antibody) demonstrated specificity of the antibody for the affinity-purified pupal MBL. The immune pupal hemolymph contained 21 microg of MBL per ml of hemolymph. The amino acid composition of the purified pupal MBL was determined with high amounts of arginine and histidine detected. The presence of MBL in insect pupae has not before been reported and could be important in pupal innate immunity to bacterial infection.

Ureaplasma urealyticum binds mannose-binding lectin.

Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.

Antiviral activity against human immunodeficiency virus-1 in vitro by myristoylated-peptide from Heliothis virescens.

An insect antiviral compound was purified from Heliothis virescens larval hemolymph by gel-filtration high pressure liquid chromatography (HPLC) and C-18 reverse-phase HPLC and its structure was determined by mass spectrometry. The antiviral compound is an N-myristoylated-peptide containing six amino acids with calculated molecular weight of 916 Da. The N-terminus contains the fatty acid myristoyl, and the C-terminus contains histidine with two methyl groups giving the histidine a permanent positive charge. The remainder of the compound is essentially non-polar. The structure of the compound corresponds with the 'myristate plus basic' motif expressed by certain viral proteins in their binding to the cytoplasmic side of the plasma membrane to initiate viral assembly and budding from a host cell. The insect antiviral compound may inhibit viral assembly and/or budding of viruses from host cells that could include the human immunodeficiency virus-1 (HIV-1) and herpes simplex virus-1 that use this motif for exit from a host cell. Using the formazan assay, the myristoylated-peptide was effective against HIV-1, with a nine times increase in the viability and protection in vitro of treated CEM-SS cells when compared with infected but untreated control cells.

Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes.

The channel catfish (Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment. Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila. The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities. Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish. Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid. The granule extracts were further purified by gel filtration chromatography. Bactericidal assays against the two fish pathogens and SDS-PAGE analysis were done on the isolated antimicrobial factor. Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1. Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor.

Ureaplasma in lung. 1. Localization by in situ hybridization in a mouse model.

Ureaplasma urealyticum is a common inhabitant of mucosal surfaces but is also associated with a higher incidence of pneumonia and bronchopulmonary dysplasia in preterm infants. Culture and polymerase chain reaction demonstrate high isolation rates of ureaplasma in clinical specimens documenting their presence but do not associate the organism directly with the diseased tissue. In this study, lung tissue samples from newborn mice inoculated intranasally with U. urealyticum were used to develop an in situ hybridization (ISH) test for the organism. In situ hybridization allows the localization of gene expression for visualization within the context of tissue morphology. New techniques which use biotinyl-tyramide based signal amplification have been able to greatly enhance the sensitivity of ISH. Using the Dako GenPoint Catalyzed Signal Amplification system to detect a biotinylated DNA probe specific for an internal nucleotide sequence within the urease gene of U. urealyticum, the organism was detected within the infected murine lung tissues. Electron microscopy was used to verify the presence of the organisms in the positive ISH areas. The ISH procedure developed in this study can be used to analyze the presence of ureaplasma in human neonatal lung tissue with the corresponding histopathology.

Ureaplasma in lung. 2. Association with bronchopulmonary dysplasia in premature newborns.

Infants with Ureaplasma urealyticum in the lower respiratory tract are at risk for chronic lung disease (CLD) or bronchopulmonary dysplasia (BPD) but causality has been difficult to prove. The goal of this study was to identify ureaplasma in human neonatal lung tissue using the in situ hybridization (ISH) procedure described in Part 1 (Exp. Mol. Pathol., in press) of this report. By correlating their presence with the histopathologic findings, it may be possible to provide further evidence of the pathogenicity of ureaplasmas and their association with BPD. Lung autopsy tissue from seven infants with positive cultures and seven infants with negative cultures for ureaplasma were included in the study. All culture-positive infants were positive for ureaplasma on ISH and all had histopathologic evidence of BPD. Two of the seven infants with negative cultures were positive for ureaplasma with ISH. Of interest, these two infants were also found to have BPD at autopsy. The other five infants with negative cultures were also negative for ureaplasma on ISH and had no evidence of BPD. This study correlates the presence of U. urealyticum by ISH with the finding of BPD on histopathologic evaluation and provides evidence that it has a role in the development of CLD.

Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae.

Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17965.9+/-5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56-83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted alpha-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic alpha-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240-340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.