Behavior of Escherichia coli K antigens K88ab, K88ac, and K88ad in immunoelectrophoresis, double diffusion, and hemagglutination.

Porcine enteropathogenic Escherichia coli strains were found to possess a variant of the K88 antigen provisionally termed K88ad. We propose to include this antigen into the international E. coli typing scheme. Ultrasonic extracts of field strains of E. coli possessing the K88ab, K88ac, or K88ad antigen and their E. coli K-12 K88+ transconjugants showed a specific K88 precipitation line in immunoelectrophoresis and double diffusion only when grown at 37 degrees C, but not when grown at 18 degrees C. By using agarose gels, K88ab, K88ac, and K88ad antigens showed anodic mobility in immunoelectrophoresis. When using Difco Noble agar gels, K88ad was not mobile or anodic, K88ab was cathodic; K88ac of 17 strains was cathodic and of 24 strains was anodic. The immunoelectrophoretic behavior of a K88 antigen (K88ab, K88ac, or K88ad) did not alter after transfer of the corresponding plasmid to E. coli K-12. Anodic and cathodic K88ac antigens could not be distinguished serologically. The differences between the results obtained in Noble agar gels and agarose gels are due to electro-endosmotic flow. We describe a procedure which increases the detection level of K88+ transconjugants in a mating mixture. It is based on the specific mannose-resistant attachment of K88+ cells to guinea pig erythrocytes.     

Monoclonal antibodies to K88ab, K88ac and K88ad fimbriae from enterotoxigenic Escherichia coli

K88ab, K88ac and K88ad fimbriae derived from enterotoxigenic Escherichia coli strains involved in porcine colibacillosis were used to immunize BALB/c mice. Several hybridomas secreting monoclonal antibodies (MAbs) against the three intact K88 fimbriae subtypes were produced by fusion of spleen cells from these mice with P3-X63-Ag8.653 myeloma cells. Hybridomas producing MAbs with affinity for all 3 E. coli K88 subtypes proved to be the most frequent (248/303), but subtype-specific monoclonals (39/303) as well as MAbs reacting with two but not with the third subtype (16/303) were also produced. The antibody-containing culture supernatants from 71 selected hybridomas were characterized by enzyme-linked immunosorbent assay (ELISA) titrations, ELISA inhibition experiments and further examined by immunoblotting. Derivation of several MAbs specific for one of the E. coli fimbrial antigens, K88ab, K88ac or K88ad, was of interest in view of the extensive sequence homology in their primary structures. Specific binding of the MAbs to fimbriae on the surface of K88-positive E. coli strains was indicated by agglutination tests and visualized by immuno gold labeling and electron microscopy. The present MAbs against K88 fimbriae have potential veterinary applications for diagnosis and treatment of porcine colibacillosis. Preliminary results indicate the therapeutic value of oral administration of murine ascitic fluid containing anti-K88 MAbs to piglets experimentally infected with E. coli K88.     

Sequence-based typing reveals a novel DLA-88 allele, DLA-88*04501, in a beagle family

The dog is an important animal model for solid organ as well as stem cell allo transplantation. Methods such as cellular and serological typing and more recently sequence-based typing (SBT) have been used to discriminate tissue antigen disparity of donor and recipient. We applied SBT for the canine class I (DLA-88) and class II (DLA-DRB1) genes in beagle families prior stem cell transplantation. A novel DLA-88 (DLA-88*04501) allele in combination with a DLA-DRB1*01901 allele was found. Sequence comparison of exons 2 and 3 of the novel allele revealed most sequence identity to the DLA-88*01301 allele (96.15% identity at the nucleotide and 90.65% identity at the protein level).     

Different pig phenotypes affect adherence of Escherichia coli to jejunal brush borders by K88ab, K88ac, or K88ad antigen.

At least five different porcine phenotypes were distinguished with the three serological variants of the K88 antigen in the brush border adhesion test. Pigs of one phenotype (A) are susceptible to adherence of all three variants, pigs of three phenotypes are susceptible to only two (B and C) or one (D) of the K88 variants, and pigs of one phenotype (E) are entirely resistant to adhesion of K88 antigen did not interfere with the adhesion of K88ab- or K88ac-positive Escherichia coli, whereas in most cases K88ab and K88ac antigen completely blocked the adhesion of K88ad-positive E. coli. Likewise, K88ab antigen blocked the adhesion of K88ac-producing E. coli to both type A and type B brush borders, and vice versa.     

An improved method for dog leukocyte antigen 88 typing and two new major histocompatibility complex class I alleles, DLA-88*01101 and DLA-88*01201

Development of preclinical dog models of solid organ and hematopoietic transplantation is critically dependent upon characterization of the polymorphic major histocompatibility complex class I and class II loci. While the class II alleles are easily typed as the polymorphic positions reside on a single exon, typing the class I locus is tedious. We have improved the class I typing method by designing improved primers and adopting alternative DNA amplification and cloning reagents that circumvent the use of radioactivity and the need for the single-stranded conformation polymorphism gels. The method is reliable in typing dogs for the class I dog leukocyte antigen (DLA)-88 locus, and through its use, we describe here two new alleles DLA-88*01101 and DLA-88*01201.     

Mucus clearance, MyD88-dependent and MyD88-independent immunity modulate lung susceptibility to spontaneous bacterial infection and inflammation

It has been postulated that mucus stasis is central to the pathogenesis of obstructive lung diseases. In Scnn1b-transgenic (Scnn1b-Tg⁺) mice, airway-targeted overexpression of the epithelial Na⁺ channel β subunit causes airway surface dehydration, which results in mucus stasis and inflammation. Bronchoalveolar lavage from neonatal Scnn1b-Tg⁺ mice, but not wild-type littermates, contained increased mucus, bacteria, and neutrophils, which declined with age. Scnn1b-Tg⁺ mice lung bacterial flora included environmental and oropharyngeal species, suggesting inhalation and/or aspiration as routes of entry. Genetic deletion of the Toll–interleukin-1 receptor adapter molecule MyD88 in Scnn1b-Tg⁺ mice did not modify airway mucus obstruction, but caused defective neutrophil recruitment and increased bacterial infection, which persisted into adulthood. Scnn1b-Tg⁺ mice derived into germ-free conditions exhibited mucus obstruction similar to conventional Scnn1b-Tg⁺ mice and sterile inflammation. Collectively, these data suggest that dehydration-induced mucus stasis promotes infection, compounds defects in other immune mechanisms, and alone is sufficient to trigger airway inflammation.     

2008: A MyD88 O, DC

In this issue of Immunity, Hou et al. (2008) describe the generation of mice that selectively lack the adaptor protein MyD88 in dendritic cells (DCs). These mice demonstrate that the requirement for Toll-like receptor (TLR) signaling in DCs is dependent on the physical form of the TLR ligand.     

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA and gene in Japanese flounder, Paralichthys olivaceus

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily signaling involves myeloid differentiation factor 88 (MyD88) that acts as an important adapter protein. A Japanese flounder (Paralichthys olivaceus) MyD88 (jfMyD88) cDNA and gene were cloned, and found to have lengths of 1.5 and 3.01 kb, respectively. The ORF encodes 285 amino acids that contain a death domain and a Toll/IL-1 receptor domain. The gene is composed of 5 exons and 4 introns. The jfMyD88 gene is highly expressed in organs involved in immune functions, including the gills, intestines, kidney, skin and spleen. Three days after a fish was infected with Edwardsiella tarda, staining with anti-jfMyD88 polyclonal antibody revealed an increased population of MyD88-positive cells in the kidney and spleen. These results imply that MyD88 has an important role in the innate immune system in Japanese flounder.     

LFP-20, a porcine lactoferrin peptide,ameliorates LPS-induced inflammation via the MyD88/NF-κB and MyD88/MAPK signaling pathways

LFP-20 is one of the 20 amino acid anti-microbial peptides identified in the N terminus of porcine lactoferrin. Apart from its extensively studied direct anti-bacterial activity, its potential as an activator of immune-related cellular functions is unknown. Therefore, this study investigated its anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated pig alveolar macrophages in vitro and systemic inflammation in an in vivo mouse model. We found that the inhibitory effects of LFP-20 on production of pro-inflammatory cytokines were independent of its LPS-binding activity. However, they were associated with NF-κB and MAPK-dependent signaling. Furthermore, LFP-20 might directly influence MyD88 levels to block its interaction with NF-κB and MAPK-dependent signaling molecules that might alter LPS-mediated inflammatory responses in activated macrophages. Taken together, our data indicated that LFP-20 prevents the LPS-induced inflammatory response by inhibiting MyD88/NF-κB and MyD88/MAPK signaling pathways, and sheds light on the potential use of LFP-20 in the therapy of LPS-mediated sepsis.     

Characterization of the Binding Specificity of K88ac and K88ad Fimbriae of Enterotoxigenic Escherichia coli by Constructing K88ac/K88ad Chimeric FaeG Major Subunits

Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) fimbriae are the major cause of diarrhea in young pigs. Three antigenic variants of K88 fimbriae (K88ab, K88ac, and K88ad) have been identified among porcine ETEC strains. Each K88 fimbrial variant shows a unique pattern in binding to different receptors on porcine enterocytes. Such variant specificity in fimbrial binding is believed to be controlled by the major subunit (FaeG) of the K88 fimbriae, because the genes coding for the only other fimbrial subunit are identical among the three variants. Uniqueness in binding to host receptors may be responsible for differences in the virulence levels of porcine diarrhea disease caused by K88 ETEC strains. To better understand the relationships between the structure of FaeG proteins and fimbrial binding function, and perhaps virulence in disease, we constructed and expressed various K88ac/K88ad faeG gene chimeras and characterized the binding activity of each K88 chimeric fimbria. After verifying biosynthesis of the chimeric fimbriae, we examined their binding specificities in bacterial adherence assays by using porcine brush border vesicles that are specific to either the K88ac or K88ad fimbria. Results showed that each fimbria switched binding specificity to that of the reciprocal type when a peptide comprising amino acids 125 to 163 was exchanged with that of its counterpart. Substitutions of a single amino acid within this region negatively affected the binding capacity of each fimbria. These data indicate that the peptide including amino acids 125 to 163 of the FaeG subunit is essential for K88 variant-specific binding.     

Enterotoxins, O-groups, and K88 Antigen in Escherichia coli from Neonatal Piglets With and Without Diarrhea

In a comparison between piglets (1-7 days old) with diarrhea and healthy piglets of the same age and 1 to 8 weeks old, 810 intestinal strains of Escherichia coli from 81 piglets from as many different herds in Sweden were investigated with regard to O-group, enterotoxicity, and possession of K88 antigen. A clear difference was found between the E. coli isolates from piglets with diarrhea and from representatives of healthy herds without diarrhea, with regard to (i) the homogeneity of strains in individual pigs and (ii) the distribution of O-groups, K88 antigen, and frequency of enterotoxicity. Strains from piglets with diarrhea showed a high frequency of O-group 149 (53%), enterotoxicity (61%), and K88 antigen (56%), while not more than 3% of the strains from healthy piglets of the same age harbored any of these characteristics. Of the isolates in O-groups 8, 64, and 115, 36% were enterotoxigenic. The corresponding data for O-group 149 and nontypable strains were 96 and 1%, respectively. Furthermore, K88 antigen was only found in O-groups 8 and 149. In O-group 149, 96% of the strains (n = 167) produced the K88 antigen as well as heat-labile enterotoxin. In contrast, strains producing heat-stable enterotoxin were mainly found in O-groups 8, 9, 64, 115, and 141. There was a significant difference in the frequencies of E. coli strains producing heat-labile enterotoxin between piglets with diarrhea and those without, whereas the pathogenic role of heat-stable enterotoxin-producing strains was less apparent.     

PG490-88, a derivative of triptolide, blocks bleomycin-induced lung fibrosis

In this study we evaluate the antifibrotic properties of PG-490-88, a water-soluble derivative of triptolide. Triptolide is an oxygenated diterpene that is derived from a traditional Chinese herb that has potent immunosuppressive and antitumor activity. We used the intratracheal bleomycin mouse model and found that PG490-88 inhibits fibrosis in the bleomycin group when given the same day or 5 days after bleomycin. PG490-88 also markedly reduced the number of myofibroblasts in the bleomycin treatment group. An enzyme-linked immunosorbent assay of transforming growth factor (TGF)-beta in the bronchoalveolar lavage fluid showed a significant decrease in TGF-beta in the PG490-88-treated groups compared to the bleomycin-treated group. Additionally, triptolide blocked bleomycin-induced increase in TGF-beta mRNA in cultured normal human lung fibroblasts. The efficacy of PG490-88 when administered late after bleomycin installation suggests a potential role in the treatment of idiopathic pulmonary fibrosis.