Bilateral acute pyogenic conjunctivitis with iritis induced by unilateral topical application of bacterial peptidoglycan muramyl dipeptide in adult rabbits.

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation.

Expression of enterovirus 70 capsid protein VP1 in Escherichia coli.

The VP1 gene of enterovirus 70 (EV70) possesses a large number of Escherichia coli low-usage codons (11.0%) and a bacterial ribosome binding site complementary sequence (RBSCS) 5'-UGUCUCCUUUUC-3' flanking the codon 139. Plasmids containing EV70 cDNA encoding the full-length VP1 failed to express in E. coli (BL21(DE3), Rosetta 2(DE3) or Rosetta (DE3)pLysS). High expression (>8% of total protein) of recombinant VP1 (rVP1m) in E. coli required engineering of the encoding cDNA (conserved modification of the native cDNA) by simultaneous substitution of a rare-codon cluster located between codons 103 and 132, and replacement of the RBSCS-TCCTTT sequence. The rare-codon frequencies of the cDNAs encoding VP1 non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) are similar (10.9 and 11.2%, respectively). However, in E. coli, high expression of recombinant C170 (rC170) required no modification of the native cDNA whereas high expression of recombinant N138 (rN138m) required minimal synonymous substitution of the above rare-codon cluster. The rare-codon cluster of EV70 VP1 gene has five least-usage arginine codons (AGG/AGA) and three tandem rare-codon pairs (AGGAGG, CUAAGG, and AGACUA). Our results suggest that the rare-codon cluster (its rare codon arrangement per se and/or its related mRNA secondary structure(s)) and the RBSCS in EV70 VP1 gene, not the rare-codon frequency, constitute the key elements that suppress its expression in E. coli.

Stereo-isomer specific induction of renal cell apoptosis by synthetic muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine).

The cytotoxicity of bacterial cell wall components, muramyl dipeptide (synthetic N-acetylmuramyl-L-alanyl-D-isoglutamine; L,D-MDP) and lipopolysaccharide (LPS), was investigated in several kidney cell lines. MDP and LPS were toxic to rabbit and monkey kidney cells, MDP was toxic to canine kidney cells, but not to human or porcine kidney cells. Notably, L,D-MDP was >100-fold more cytotoxic/microg than the D,D-MDP and L,L-MDP, as well as LPS. L,D-MDP and analogs containing L,D-MDP were the most widely cytotoxic of the MDP tested. The MDP-induced cytotoxicity was characterized as apoptosis by DAPI staining and DNA laddering. The acute rabbit kidney (RK13) cell apoptosis (cell death in < 5 h) induced by apical or basal application of MDP was associated with glutamate (Glu) release, decreased gamma-glutamyltranspeptidase (GGT) and acidosis and was suppressed by Indomethacin, Naproxen and Curcumin. The cytotoxic activity of L,D-MDP was decreased significantly by 24 h incubation in human sera. Aged (> 2 year-old) rabbits that apparently failed to quickly clear and excrete a uveitogenic dose of MDP within 24 h died in I week. The results indicate that minute amounts (5 ng/ml) of MDP containing L-alanyl-D-isoglutamine can induce renal cell apoptosis in vitro and support MDP-induced kidney cytotoxicity in rabbits. Also, the results indicate that MDP in sera can be detected utilizing the RK13 cell bioassay and that failure to rapidly clear and excrete L,D-MDP is associated with uveitis and death in aged rabbits.