Annika Weber

Elevated extrahepatic expression and secretion of mammary-associated serum amyloid A 3 (M-SAA3) into colostrum.

Mammary-associated serum amyloid A 3 (M-SAA3) was secreted at highly elevated levels in bovine, equine and ovine colostrum and found at lower levels in milk 4 days postparturition. N-terminal sequencing of the mature M-SAA3 protein from all the three species revealed a conserved four amino acid motif (TFLK) within the first eight residues. This motif has not been reported to be present in any of the hepatically-produced acute phase SAA (A-SAA) isoforms. Cloning of the bovine M-Saa3 cDNA from mammary gland epithelial cells revealed an open reading frame that encoded a precursor protein of 131 amino acids which included an 18 amino acid signal peptide. The predicted 113 residue mature M-SAA3 protein had a theoretical molecular mass of 12,826Da that corresponded with the observed 12.8kDa molecular mass obtained for M-SAA3 in immunoblot analysis. The high abundance of this extrahepatically produced SAA3 isoform in the colostrum of healthy animals suggests that M-SAA3 may play an important functional role associated with newborn adaptation to extrauterine life and possibly mammary tissue remodeling.

A monoclonal antibody sandwich immunoassay for serum amyloid A (SAA) protein

An antibody sandwich immunoassay using two purified rat monoclonal antibodies to human serum amyloid A was developed and used to measure serum amyloid A in human serum. The assay was specific, sensitive, reproducible, and reliable and does not require denaturation of the specimen prior to assay. Serum amyloid A purified by hydrophobic interaction chromatography of acute phase human serum afforded a reliable standard for the assay. A significant (r = 0.69) correlation for SAA and C reactive protein values was found for 180 patient samples analyzed.

Human serum amyloid A3 peptide enhances intestinal MUC3 expression and inhibits EPEC adherence.

We previously determined that the N-terminal region of bovine mammary-associated serum amyloid A3 (M-SAA3) increased intestinal mucin MUC3 levels in HT29 human intestinal cells by approximately 2.5-fold, relative to untreated cells. This study shows that the human M-SAA3 N-terminal peptide further enhances MUC3 transcript levels by approximately 4.3-fold in these cells (p<0.02), implicating a species-specific interaction. Furthermore, immunofluorescence and immunoblot analysis using a MUC3-specific monoclonal antibody confirms that the human M-SAA3 peptide stimulates MUC3 protein expression and secretion by the HT29 cells. More importantly, pretreatment of the cells with the peptide causes a subsequent 73% decrease in the adherence of enteropathogenic Escherichia coli (EPEC) to these cells, relative to untreated cells (p<0.01). The intestinal mucin MUC3 has been shown to provide a protective barrier in the gut and inhibit adherence of pathogens to the gut wall. Therefore, a means to increase MUC3 protein expression by a colostrum-associated peptide or protein may be a highly effective prophylactic treatment for the prevention of gastrointestinal diseases such as necrotizing enterocolitis and infectious diarrhea.

The conserved TFLK motif of mammary-associated serum amyloid A3 is responsible for up-regulation of intestinal MUC3 mucin expression in vitro.

In various mammalian species, an isoform of serum amyloid A is secreted at high concentrations into colostrum. A conserved four-amino-acid motif (TFLK) is contained within the first eight N-terminal amino acid residues of this mammary-associated serum amyloid A isoform 3 (M-SAA3). Peptides derived from the bovine N-terminal amino acid sequence of M-SAA3 were produced and added to cell culture medium of HT29 cells to study the effects on intestinal mucin gene expression. HT29 cells were grown to enhance expression of either MUC2 or MUC3 intestinal mucins. After incubation, total RNA was isolated for Northern blot analyses using MUC2 or MUC3 mucin cDNA probes. Signals were detected by autoradiography with mRNA levels expressed relative to 28S rRNA. The 10-mer peptides containing the intact TFLK-motif or a TFLK 4-mer peptide increased MUC3 mRNA expression compared with control cells (p < 0.05). There was no effect of these peptides on MUC2 mRNA expression. Cells that were incubated with 10-mer N-terminal derived peptides containing a scrambled TFLK motif, with all 10 amino acid residues scrambled or derived from a C-terminal region of M-SAA3, did not show increased MUC3 expression. Inhibition of enteropathogenic Escherichia coli strain E2348/69 adhesion to HT29 cells grown to enhance MUC3 expression was reduced by a similar amount when either peptides containing the intact TFLK motif or probiotic microbes were added to cell culture medium compared with control cells. M-SAA3 is a bioactive peptide secreted into colostrums that can up-regulate mucin expression and thereby may enhance innate protective mechanisms that limit access of deleterious microbes to intestinal mucosal epithelial cells in the postparturition period.

Evaluation of colostrum-derived human mammary-associated serum amyloid A3 (M-SAA3) protein and peptide derivatives for the prevention of enteric infection: in vitro and in murine models of intestinal disease

In vitro experiments confirmed that a 10-mer peptide derived from human mammary-associated serum amyloid A3 (M-SAA3) protected intestinal epithelial cells from enteropathogenic Escherichia coli (EPEC) adherence. The entire 42-mer human M-SAA3 protein was even more effective, reducing EPEC binding by 72% relative to untreated cells (P<0.05), compared with 25% and 57% reductions for the human 10-mer and Lactobacillus GG, respectively. However, none of the M-SAA3 peptides reduced Salmonella invasion in vitro (P>0.05). Each of the M-SAA3 10-mer peptides and the 42-mer was then administered orally to mice at 500 mug day(-1) for 4 days before deliberate infection with either Citrobacter rodentium (mouse model of EPEC) or Salmonella Typhimurium. None of the peptides protected against Salmonella infection and the 42-mer may even increase infection, as there was a trend towards increased Salmonella counts in the liver and small intestine in 42-mer-treated mice compared with those in sodium acetate-treated control mice. Citrobacter counts were reduced in the caecum of mice administered the 42-mer relative to a scrambled 10-mer (P<0.05), but not compared with the sodium acetate control and no reductions were observed in the faeces or colon. Overall, although promising anti-infective activity was demonstrated in vitro, neither the 42-mer M-SAA3 protein nor a 10-mer peptide derivative prevented enteric infection in the animal models tested.

Creatinine inhibits bacterial replication

Widespread antibiotic use has resulted in increased frequency of clinically important bacteria acquiring single or multiple antibiotic resistance.1,2 Even antibiotic therapies for relatively trivial afflictions, such as acne,3 have promoted development of microbial antibiotic resistance. The availability of non-prescription antibiotics in some areas has also resulted in improper and/or irrational self-medication, further exacerbating this problem.4,5 New antibacterial agents with broad-spectrum impact against both Gram-positive6 and Gram-negative7 species, as well as against drug-resistant strains such as methicillinresistant Staphylococcus aureus8 are needed for wound care and to treat topical and dermatological infections.9 A chance observation in our laboratory revealed that creatinine (CRN; creatinine hydrochloride, CRN-HCl) halted the growth of bacteria on nutrient agar plates. CRN is the naturally occurring breakdown product of creatine phosphate, a high-energy molecule used to store and then donate, a high-energy phosphate to ADP for the synthesis of ATP in metabolism. Occurring normally in human blood at concentrations ranging approximately between 50–100mM and in urine at slightly higher levels, CRN is accepted to be a naturally produced inert waste product with no active function,10 although a recently published study has challenged this dogma.11 We characterized the ability of CRN-HCl to inhibit the growth of a wide array of bacterial species, including methicillinresistant Staphylococcus aureus. CRN-HCl (Sigma-Aldrich; St Louis, MO, USA; F.W.1⁄4149.6) was evaluated for its ability to inhibit the growth of different bacteria. Bacterial cultures were propagated in LBG (LB broth, Miller; Fisher Scientific; Fair Lawn, NJ, USA) supplemented with 1% w/v glucose) either in liquid or agar (BD Bacto Agar; Becton Dickinson, Sparks, MD, USA) format. Overnight cultures of Escherichia coli and S. aureus were diluted in fresh LBG to 1–5 105 colony-forming units (c.f.u.) per ml, aliquoted to 2 ml tube cultures, to each of which was added CRN-HCl in increasing 5 mM increments from 0–50 mM. After shaking at 250 r.p.m. at 37 1C for 18 h, the OD at 600 nm was recorded. E. coli was inhibited from growth in 40 mM (5.98 mg ml 1) CRN-HCl, while S. aureus growth was inhibited at a concentration of 15 mM (2.24 mg ml 1) CRN-HCl (Figure 1a). These values were taken as a measurement of the MIC for CRN-HCl under these specific assay conditions. A similar experiment was performed using a 96-well microtiter plate format in which triplicate wells were filled with 100ul total volume of E. coli or S. aureus (final concentration of 1– 5 105 c.f.u. ml 1) in LBG containing increasing concentrations of CRN-HCl. Following 18 h stationary incubation at 37 1C, wells were examined visually to ascertain which wells appeared turbid versus clear, then the plate was assayed using an ELISA plate reader (ELX800; Bio-Tek Instruments, Winooski, VT, USA) using a 490 nm wavelength (Figures 1b and c). This format confirmed the results from the tube cultures, demonstrating the inhibition of the growth of E. coli and S. aureus at 40 and 15 mM CRN-HCl, respectively. Using this microtiter plate format, MIC values for four other bacterial species were established: Brevibacterium linens (MIC1⁄410 mM), Bacillus subtilis (MIC1⁄420 mM), Pseudomonas aeruginosa (20 mM) and Streptococcus pyogenes (MIC1⁄420 mM; data not shown). Drug-resistant bacterial strains and other bacterial species were assayed for sensitivity to inhibition of replication by CRN-HCl using a disc diffusion assay. Assays were performed as described12 with the following modifications. Twenty-five microliters of 2 M CRN-HCl in water was added to 30 mg of a powdered carrier (Eridex; Cargill Inc., Cedar Rapids, IA, USA) and stirred into a thickened slurry in order to apply the maximum amount of CRN-HCl on the disc. Fifty microliters of the slurry containing 5 mg CRN-HCl were applied to 6 mm diameter sterile dry paper discs (Whatman no. 3 filter paper; Whatman, Piscataway, NJ, USA) that were then inverted onto LBG agar plates containing test bacteria. The LBG agar plates were prepared 15 min before use by spreading the test bacteria (diluted in phosphate-buffered saline to 1–5 105 c.f.u. ml 1 from overnight cultures) to the plate with a sterile cotton swab. The plates with discs were incubated for 15 h at the temperature appropriate for the particular bacterial species. Clear zones around the discs, indicative of growth inhibition, were measured. As a point of comparison, gentamicin-impregnated discs (GM-10; Becton Dickinson) were tested. For all bacteria but drug-resistant and anaerobic bacteria, for which assays were repeated twice, discs were tested in triplicate on two different days and zones of inhibition were recorded as the average of these six measurements. Variation was p2 mm. Results for the diverse bacterial species assayed using this approach are shown in Table 1. All bacteria tested in this manner, including drug-resistant strains,

Construction and use of a template block for radial immunodiffusion.

Details for design and construction of template blocks for the increased sensitivity of radial immunodiffusion (RID) assays are described. The sensitivity of the template RID was 10-fold higher for quantifying albumin and 5-fold higher for IgG than the conventional RID with wells cut into the agarose. The system described in this communication resulted from the unit which provided the most consistent results from a variety of trials with different block thicknesses, chamber sizes and depths, and chamber arrangements.