Defensin D5

Backgrounds and Aims:Klebsiella pneumoniae represents the most common opportunistic pathogen contributing to Klebsiella pneumonia in hospital-acquired infections. Klebsiella pneumonia has a rapidly progressive clinical course and multi-drug resistant (MDR). Identification of the effective biochemical markers is crucial for improving early diagnosis and treatment of Klebsiella pneumonia. The aims of our study are to 1) investigate the expression of β-Defensin-2(rβD2), IL-22, IL-22R1 and IL-10R2 in Klebsiella pneumonia-infected rats and 2) their association with the histological grades of Klebsiella pneumonia.Methods and Materials: Fifty specific pathogen free (SPF) male SD rats were randomly divided into two groups: control group (treated with normal saline) and pneumonia group (treated with K. pneumoniae). All animals were sacrificed 1 h, 12 h, 1 d, 3 d, 5 d post infection. The severity and property of pneumonia was evaluated by histopathologic observation and pathogen identification. The mRNA expression of rβD2, IL-22, IL-22R1 and IL-10R2 was measured by RT-qPCR assay. The expression of rβD2 in rat lung tissue was determined by Western blot analysis, and the level of IL-22 in rat serum was determined by ELISA.Results: Histopathologic examination and bacterial counting of lung tissues confirmed the successful establishment of rat pneumonia model. The gene expression of rβD2, IL-22, IL-22R1 and IL-10R2 in pneumonia rats were significantly higher than those in healthy control mice (P < 0.05). The expression of rβD2 was correlated with histological grades of Klebsiella pneumonia and the level of IL-22. RT-qPCR results showed that the peak expression of IL-22R1 appeared earlier than IL-10R2 in rat pneumonia model.Conclusions: The expression of rβD2 and IL-22 was increased significantly at early stage in rat Klebsiella pneumonia model, suggesting that IL-22 and rβD2 might serve as potential biomarkers for the early diagnosis of Klebsiella pneumonia.

We describe here a novel method for generation of yeast-secreted, in vivo biotinylated recombinant antibodies, or biobodies. Biobodies are secreted by diploid yeast resulting from the fusion of two haploid yeast of opposite mating type. One yeast carries a cDNA encoding an antibody recognition sequence fused to an IgA1 hinge and a biotin acceptor site (BCCP) at the C-terminus; the other carries a cDNA encoding an E. coli biotin ligase (BirA) fused to KEX2 golgi-localization sequences, so that BirA can catalyze the biotin transfer to the recognition sequence-fused BCCP within the yeast secretory compartment. We illustrate this technology with biobodies against HE4, a biomarker for ovarian carcinoma. Anti-HE4 biobodies were derived from clones or pools of anti-HE4-specific yeast-display scFv, constituting respectively monoclonal (mBb) or polyclonal (pBb) biobodies. Anti-HE4 biobodies were secreted directly biotinylated thus bound to labeled-streptavidin and streptavidin-coated surfaces without Ni-purification. Anti-HE4 biobodies demonstrated specificity and sensitivity by ELISA assays, flow cytometry analysis and Western blots prior to any maturation; dissociation equilibrium constants as measured by surface plasmon resonance sensor were of K(d)=4.8 x 10(-9) M and K(d)=5.1 x 10(-9) M before and after Ni-purification respectively. Thus, yeast mating permits cost-effective generation of biotinylated recombinant antibodies of high affinity.

Lingual antimicrobial peptide (LAP) belongs to the beta-defensin family in cattle and is found in bovine milk. However, it is unclear whether LAP is involved in the early immune response to mammary infection. The aim of the study was to investigate the changes of LAP concentration in milk after intramammary challenge with lipopolysaccharide (LPS), the gram-negative bacteria cell membrane component, in dairy cows. Milk was collected before and after LPS or phosphate-buffered saline (control) challenge every hour for 12 h on d 0 and twice daily from d 1 to 7. Somatic cell count (SCC), LAP concentration, and lactoperoxidase (LPO) activity in the milk were measured. Somatic cell count started to increase at 2 h postchallenge and remained high until d 5 (694 +/- 187 x 10(3 )to >1,000 +/- 0 x 10(3) cells/mL at d 0; >1,000 +/- 0 x 10(3) cells/mL at d 1 to 3; 684 +/- 194 x 10(3 )to 829 +/- 108 x 10(3 )cells/mL at d 4; 527 +/- 197 x 10(3 )to 656 +/- 145 x 10(3 )cells/mL at d 5). Somatic cell count increased in the control cows, although the levels were lower compared with those in the LPS challenge group. The LAP concentration in milk increased significantly at 2 h post-LPS-challenge and was maintained at high levels until d 2 (8.6 +/- 0.6 to 17.5 +/- 2.3 nM). In the control cow infused with phosphate-buffered saline, there was no increase of LAP concentration in milk (5.1 +/- 0.6 to 7.2 +/- 0.8 nM). Increase of LPO activity in the milk was observed at 6 h after LPS challenge and continued until d 3 (4.7 +/- 0.3 to 9.4 +/- 1.1 U). No increase of LPO activity was observed in the milk of control cows. The increase and subsequent decrease in LAP concentration after LPS challenge occurred earlier than those of LPO activity. In multiparous cows with LPS infusion, there was a significantly negative relationship between the days leading to the basal levels in LAP concentration and LPO activity (r = -0.75). These results suggest that LPS induces secretion of LAP into milk within hours and that LPO may have a synergistic antimicrobial function with LAP in mammary glands of dairy cows.