Efficacy of a single oral dose of a live bivalent E. coli vaccine against post-weaning diarrhea due to F4 and F18-positive enterotoxigenic E. coli.

F4- and F18-positive enterotoxigenic E. coli strains (F4-ETEC and F18-ETEC) are important causes of post-weaning diarrhea (PWD) in pigs. F4 (antigenic variant ac) and F18 (ab and ac) fimbriae are major antigens that play an important role in the early stages of infection. Herein, the efficacy of a live oral vaccine consisting of two non-pathogenic E. coli strains, one F4ac- and one F18ac-positive, was evaluated using F4ac-ETEC and F18ab-ETEC challenge models. A randomized, masked, placebo-controlled, block design, parallel-group confirmatory study with two different vaccination-challenge intervals (7 and 21 days) was conducted for each challenge model. The vaccine was administered in one dose, to ≥18-day-old piglets via drinking water. Efficacy was assessed by evaluating diarrhea, clinical observations, weight gain and fecal shedding of F4-ETEC or F18-ETEC. Anti-F4 and anti-F18 immunoglobulins in blood were measured. The vaccination resulted in significant reductions in clinical PWD and fecal shedding of F4-ETEC and F18-ETEC after the 7- and 21-day-post-vaccination heterologous challenges, except for after the 21-day-post-vaccination F4-ETEC challenge, when the clinical PWD was too mild to demonstrate efficacy. A significant reduction of mortality and weight loss by vaccination were observed following the F18-ETEC challenge. The 7-day protection was associated with induction of anti-F4 and anti-F18 IgM, whereas the 21-day protection was mainly associated with anti-F4 and anti-F18 IgA. The 7-day onset and 21-day duration of protection induced by this vaccine administered once in drinking water to pigs of at least 18days of age were confirmed by protection against F4-ETEC and F18-ETEC, and induction of F4 and F18-specific immunity. Cross protection of the vaccine against F18ab-E. coli was demonstrated for both the 7- and 21-day F18-ETEC challenges.

In vitro activity of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine against multidrug-resistant hepatitis B virus mutants.

The susceptibilities of drug-resistant hepatitis B virus (HBV) mutants to lamivudine, adefovir, tenofovir, entecavir, and 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]-pyrimidine (PMEO-DAPym), a novel acyclic pyrimidine analogue, were assessed in vitro. Most drug-resistant mutants, including multidrug-resistant strains, remained sensitive to tenofovir and PMEO-DAPym. Therefore, the latter molecule deserves further evaluation for the treatment of HBV infection.

Cyclooxygenase-2 expression in normal and neoplastic canine mammary cell lines.

Mammary cancer is the most common cancer in female dogs. Induction of cyclooxygenase-2 (COX-2), a key enzyme in prostaglandins (PGs) biosynthesis, has been demonstrated in various cancers in humans and dogs, including mammary cancer. The objective of this study was to investigate the expression and regulation of COX-2 in canine mammary epithelial cells. Cell lines derived from normal and neoplastic canine mammary glands were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and immunoblots, immunocytochemistry, radioimmunoassays, and a cell proliferation assay were used to study COX-2 expression and PGs production. Results showed that the neoplastic cell line CMT12 constitutively overexpressed COX-2 protein whereas other mammary cell lines expressed low to undetectable basal levels of COX-2 protein. Basal PGE(2) production was significantly higher (P < .05) in CMT12 compared to other cell lines. Levels of COX-2 protein in CMT12 decreased in a time-dependent manner with serum starvation, and PMA stimulation induced a strong time-dependent increase in COX-2 protein. Treatment of CMT12 cells with NS-398 (a specific COX-2 inhibitor) significantly blocked PGE(2) synthesis and reduced cell proliferation (P < .05). These results indicate that some neoplastic canine mammary cell lines constitutively overexpress COX-2, and that COX-2 inhibition decreases PGE(2) production and cell proliferation, supporting a role for COX-2 and PGs in canine mammary oncogenesis.

In vitro characterization of the anti-hepatitis B virus activity and cross-resistance profile of 2',3'-dideoxy-3'-fluoroguanosine.

The fluorinated guanosine analog 2',3'-dideoxy-3'-fluoroguanosine (FLG) was shown to inhibit wild-type (wt) hepatitis B virus (HBV) replication in a human hepatoma cell line permanently expressing HBV. Experiments performed in the duck model of HBV infection also showed its in vivo antiviral activity. In this study, we investigated the mechanism of inhibition of FLG on HBV replication and its profile of antiviral activity against different HBV or duck hepatitis B virus (DHBV) drug-resistant mutants. We found that FLG-triphosphate inhibits weakly the priming of the reverse transcription compared to adefovir-diphosphate in a cell-free system assay allowing the expression of an enzymatically active DHBV reverse transcriptase. It inhibits more potently wt DHBV minus-strand DNA synthesis compared to lamivudine-triphosphate and shows a similar activity compared to adefovir-diphosphate. FLG-triphosphate was most likely a competitive inhibitor of dGTP incorporation and a DNA chain terminator. In Huh7 cells transiently transfected with different HBV constructs, FLG inhibited similarly the replication of wt, lamivudine-resistant, adefovir-resistant, and lamivudine-plus-adefovir-resistant HBV mutants. These results were consistent with those obtained in the DHBV polymerase assay using the same drug-resistant polymerase mutants. In conclusion, our data provide new insights in the mechanism of action of FLG-triphosphate on HBV replication and demonstrate its inhibitory activity on drug-resistant mutant reverse transcriptases in vitro. Furthermore, our results provide the rationale for further clinical evaluation of FLG in the treatment of drug-resistant virus infection and in the setting of combination therapy to prevent or delay drug resistance.

Expression of the human immunodeficiency virus frameshift signal in a bacterial cell-free system: influence of an interaction between the ribosome and a stem-loop structure downstream from the slippery site.

A-1 frameshift event is required for expression of the pol gene when ribosomes translate the mRNA of human immunodeficiency virus type-1 (HIV-1). In this study, we inserted the frameshift region of HIV-1 (a slippery heptanucleotide motif followed by a stem-loop) in a reporter gene coding for firefly luciferase. The ability of the corresponding mRNA, generated by in vitro transcription, to be translated in an Escherichia coli cell-free extract is the first demonstration that the HIV-1 frameshift can be reproduced in a bacterial cell-free extract, providing a powerful approach for analysis of the frameshift mechanism. The responses of the frameshift signal to chloramphenicol, an inhibitor of peptide bond formation, and spectinomycin, an inhibitor of translocation, suggest that the frameshift complies with the same rules found in eukaryotic translation systems. Furthermore, when translation was performed in the presence of streptomycin and neamine, two error-inducing antibiotics, or with hyperaccurate ribosomes mutated in S12, the frameshift efficiency was increased or decreased, respectively, but only in the presence of the stem-loop, suggesting that the stem-loop can influence the frameshift through a functional interaction with the ribosomes.

Streptomycin binds to the decoding center of 16 S ribosomal RNA.

Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3' minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3' minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.

The sonography of renal columnar hypertrophy.

An enlarged Column of Bertin is a frequent cause of renal pseudotumor. In a retrospective study of the ultrasound findings in 11 patients with columnar hypertrophy, we have attempted to establish objective sonographic criteria of this entity. Columnar hypertrophy appears as a renal mass with the following characteristics: (1) indents the renal sinus laterally; (2) clearly defined from the renal sinus; (3) largest dimension of less than 3 cm; (4) continuous or contiguous with the renal cortex; and (5) Echogenicity close to that of the cortex. Two minor criteria are also presented, and the present appropriate use of the criteria is suggested.