Enhanced antiviral and opsonic activity of a human mannose-binding lectin and surfactant protein D chimera

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.     

CL-46, a novel collectin highly expressed in bovine thymus and liver

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.     

The opsonic activity of complement in sera without antibody

Путем селекции были отобраны штаммы грамотрицательных бактерий (в фазе S), суспензия которых в растворе Ringer-Locke при перфузии не задерживается изолированной печенью. Опсонизио рвание этих штаммов сывороткой, которая не содержит антител, новорожденных поросят, не получавших еще молозива, значительно увеличивает процент задерживаемых бактерий Путем инактивирования компонентов комплемента было доказано, что опсонизирующее действие зависит от функции комплемента, даже если антитела не присутствуют.     

Vitellogenin functions as a multivalent pattern recognition receptor with an opsonic activity

Background

Vitellogenin (Vg), a major reproductive protein, has been associated with infection-resistant response in fish. However, the underlying mechanisms by which Vg is involved in anti-infectious response are not understood.

Methodology/Results

By both protein-microbe interaction analysis and enzyme-linked immunosorbent assay as well as phagocytosis test, we demonstrate for the first time that fish Vg acts as a pattern recognition molecule with multiple specificities that can recognize bacteria as well as fungus rather than self components from fish, and functions as an opsonin that can enhance macrophage phagocytosis.

Conclusions

This study shows that fish Vg plays an integrative function in regulating immunity via its pleiotropic effects on both recognizing pathogen-associated molecular patterns and promoting macrophage phagocytosis. It also supports the notion that factors normally involved in control of female reproduction are associated with immunity in organisms that rely on Vg for oocyte development.     

Identification and characterization of a novel human collectin CL-K1

Collectins are a family of C-type lectins with two characteristic structures, collagen like domains and carbohydrate recognition domains. They recognize carbohydrate antigens on microorganisms and act as host-defense. Here we report the cloning and characterization of a novel collectin CL-K1. RT-PCR analyses showed CL-K1 mRNA is present in all organs. The deduced amino acid sequence and the data from immunostaining of CL-K1 cDNA expressing CHO cells revealed that CL-K1 is expressed as a secreted protein. CL-K1 is found in blood by immunoblotting and partial amino acid analyses. CL-K1 showed Ca(2+)-dependent sugar binding activity of fucose and weakly mannose but not N-acetyl-galactosamine, N-acetyl-glucosamine, or maltose, though mannose-binding lectin (MBL) containing similar amino acid motif. CL-K1 can recognize specially several bacterial saccharides due to specific sugar-binding character. Elucidation of the role of two ancestor collectins of CL-K1 and CL-L1 could lead to see the biological function of collectin family.     

The method for estimation of opsonic activity in sera of infants

Three methods for the estimation of opsonic activity in the sera of newborn children were tested. Two of them, based on the phagocytosis of opsonised bacteria labelled with radioactive phosphorus³²P as measured byin vivo blood clearance or uptake of bacteria in perfused isolated liver, were found to be unsuitable for long term dynamic study mainly because they do not permit the testing of series of samples. The third method (using isolated phagocytic cellsin vitro) permits the differentiation of the opsonic effect of complement and antibody and, furthermore, the firmness of the bond between microbes and phagocytes (which reflects the degree of opsonization) can be established. It was found that a 2-mercaptoethanol-resistant antibody, probably of the IgG type, was responsible for the opsonic activity of children's sera toEscherichia coli 083. Homologous antibody (toEscherichia coli 083) could be differentiated from beterologous antibody (toEscherichia coli 086) using the opsonic test only at low dilutions of sera. The combination of newborn piglet complement and antibody of children's sera yielded higher values of opsonic activity than either component separately.     

The mechanism of the antiviral activity of an antiviral factor of a non-interferon nature]

Antiviral factor (AF) of protein nature has been isolated from chick embryo fibroblasts infected with Venezuelan equine encephalitis virus. The suppression of virus reproduction has been observed both in homologous and heterologous cell cultures when the preparation was introduced immediately after the adsorption of the virus after pretreatment of the cell monolayer. The study has demonstrated that the antiviral effect of AF is not linked with its IFN-alpha and TNF-alpha activity. Analysis of the results obtained in this study and earlier data contained in literature suggests that infected chick embryo fibroblasts release original cytokine of non-interferon nature with antiviral activity.     

In vitro and in vivo effects of a multimerized alphas 1-casein enhancer on whey acidic protein gene promoter activity

Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit alpha s1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit whey acidic protein gene (WAP) promoter (-6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5'HS4 insulator region from the chicken beta-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5'HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5'HS4 insulator.     

Synthesis and antiviral activity of a new series of 4-isothiazolecarbonitriles

A series of 4-isothiazolecarbonitriles was synthesized and screened for in vitro antiviral activity. The effect of various substituents on the phenyl ring, as well as the substitution of the phenyl for other aromatic and heteroaromatic rings, was examined to establish the requirements for optimum activity. The most active member of the series, 3-methylthio-5-phenyl-4-isothiazolecarbonitrile, exhibited a high level of activity against enteroviruses polio 1 and ECHO 9. Preliminary studies on its mechanism of action indicated that this compound had an effect on an early event in the replication of poliovirus type 1.     

Calcium as a possible modulator of Kupffer cell phagocytic function by regulating liver-specific opsonic activity

Recently we described some properties of organ-specific serum opsonins which differentiate between liver- and spleen-specific opsonic activities, and reported that, on dialysis of serum, its liver opsonic activity is enhanced by 2- to 3-fold, whereas spleen-specific activity is reduced by 20-30% of that of control serum (Moghimi, S.M. and Patel, H.M. (1989) Biochim. Biophys. Acta 984, 379-383). This observation suggests that serum contains dialysable factors which regulate liver- as well as spleen-specific opsonic activities. Our results from EGTA-treated serum suggest that dialysable factor(s) could be divalent cations such as Ca2+, Mn2+, Mg2+ or Co2+, and among them, calcium may be the key regulatory factor for liver-specific opsonic activity. The regulatory mechanism of spleen-specific opsonic activity seems to be complex, since addition of dialysate or calcium or magnesium to the dialysed serum does not restore its activity; probably the removal of divalent cations has induced an irreversible conformational change in spleen-specific opsonin. In conclusion, we propose that the blood calcium concentration may play an important role in modulating hepatic phagocytic function by modifying liver-specific opsonic activity in serum. An increase in the physiological concentration of calcium will suppress and a decrease will enhance this opsonic activity.     

Synthesis and antiviral activity of substituted quercetins

Influenza viruses are important pathogens that cause respiratory infections in humans and animals. In addition to vaccination, antiviral drugs against influenza virus play a significant role in controlling viral infections by reducing disease progression and virus transmission. Plant derived polyphenols are associated with antioxidant activity, anti-carcinogenic, and cardio- and neuro-protective actions. Some polyphenols, such as resveratrol and epigallocatechin gallate (EGCG), showed significant anti-influenza activity in vitro and/or in vivo. Recently we showed that quercetin and isoquercetin (quercetin-3-β-d-glucoside), a glucoside form of quercetin, significantly reduced the replication of influenza viruses in vitro and in vivo (isoquercetin). The antiviral effects of isoquercetin were greater than that of quercetin with lower IC(50) values and higher in vitro therapeutic index. Thus, we investigated the synthesis and antiviral activities of various quercetin derivatives with substitution of C3, C3', and C5 hydroxyl functions with various phenolic ester, alkoxy, and aminoalkoxy moieties. Among newly synthesized compounds, quercetin-3-gallate which is structurally related to EGCG showed comparable antiviral activity against influenza virus (porcine H1N1 strain) to that of EGCG with improved in vitro therapeutic index.     

Synthesis and antiviral activity of novel aza-acyclonucleosides

We have prepared a series of novel aza-acyclonucleosides as potential antiviral agents. These compounds were prepared from diethanolamine and the desired purine or pyrimidine base via a Mitsunobu coupling. No antiviral activity was observed against either HSV-1 or HCMV.     

Examination of the bactericidal and opsonic activity of normal human serum for a mucoid and nonmucoid strain ofPseudomonas aeruginosa

The bactericidal and opsonic activity of fresh human serum (FHS) for a mucoid strain ofPseudomonas aeruginosa, 144M, and its spontaneous nonmucoid revertant, 144NM, was examined. Strain 144M was sensitive to the bactericidal activity of FHS, but strain 144NM was not. This bactericidal activity was due to the combined interaction of IgG and IgM with complement, activated through both pathways. Neither 144M nor 144NM was ingested by human polymorphonuclear leukocytes (PMNL) without FHS. Whereas maximal phagocytosis of 144M required only 5% FHS, comparable ingestion of 144NM required 25% FHS. Maximal phagocytosis of either 144M or 144NM required IgG, IgM, and complement. However, 144M required a heat-sensitive opsonic IgG, whereas 144NM required a heat-resistant IgG. Using selective absorption techniques, the targets for bactericidal and opsonic immunoglobulins on 144M and 144NM appeared to be different, suggesting that the variant 144NM had one or more altered, absent, or inaccessible cell surface components that account for differences in response to FHS and PMNL.     

Immunolocalization of a novel collectin CL-K1 in murine tissues.

We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central veins in liver, which suggests that murine CL-K1 may be mainly produced in the liver and secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.     

Nucleoside analogues exerting antiviral activity through a non-nucleoside mechanism

In analogy with maribavir [1-(beta-L-ribofuranosyl)-isopropylamino-5,6-dichlorobenzimidazole], a nucleoside analogue that acts against human cytomegalovirus (HCMV) by a non-nucleoside mechanism, here I present three other examples of classes of nucleoside analogues (i.e. bicyclic furo[2,3-d]pyrimidine as well as HEPT and TSAO derivatives) that act against either HCMV or human immunodeficiency virus (HIV) through a non-nucleoside mode of action.     

Increased stability and antiviral activity of 2'-O-phosphoglyceryl derivatives of (2'-5')oligo(adenylate)

Metabolically stable analogues of (2'-5')oligo(adenylate), (2'-5')(A)n, might constitute a new class of antiviral agents as they mimic some of the effects of interferons. 2'-O-phosphoglyceryl derivatives of (2'-5')(A)n oligomers, (2'-5')(A)n-PGro have been synthesized by chemical modification of their terminal ribose residue. Such analogues are resistant to degradation by phosphodiesterases but remain sensitive to phosphatase activity, at least in cell-free extracts. In line with its increased stability, (2'-5')(A)n-PGro has a powerful antiviral activity against an RNA virus when microinjected with micropipettes into the cytoplasm of intact cells. This antiviral activity remains transient however, possibly as a consequence of degradation in intact cells. Since (2'-5')(A)n and its derivatives do not easily cross cell membranes, their possible use in antiviral chemotherapy is tightly linked with the development of vectors suitable for their administration in vivo.