Failure to differentiate Cryptosporidium parvum from C. meleagridis based on PCR amplification of eight DNA sequences.

In order to determine the specificities of PCR-based assays used for detecting Cryptosporidium parvum DNA, eight pairs of previously described PCR primers targeting six distinct regions of the Cryptosporidium genome were evaluated for the detection of C. parvum, the agent of human cryptosporidiosis, and C. muris, C. baileyi, and C. meleagridis, three Cryptosporidium species that infect birds or mammals but are not considered to be human pathogens. The four Cryptosporidium species were divided into two groups: C. parvum and C. meleagridis, which gave the same-sized fragments with all the reactions, and C. muris and C. baileyi, which gave positive results with primer pairs targeting the 18S rRNA gene only. In addition to being genetically similar at each of the eight loci analyzed by DNA amplification, C. parvum and C. meleagridis couldn't be differentiated even after restriction enzyme digestion of the PCR products obtained from three of the target genes. This study indicates that caution should be exercised in the interpretation of data from water sample analysis performed by these methods, since a positive result does not necessarily reflect a contamination by the human pathogen C. parvum.

Localization and characterization of the RNA binding protein TLS in skin and stratified mucosa.

Translocated in liposarcoma (TLS), a member of the Ewing's sarcoma family of RNA binding proteins, is targeted to the product of RNA POL II and functions in nuclear events as well as in nuclear-cytoplasmic transport of mRNA. It has been most extensively studied in cell lines, but was identified in several rat tissues by northern blot analysis, with adipose tissue showing the highest expression followed by whole skin. This paper describes a protein with amino acid sequence homology to TLS that was isolated from bovine tongue epithelium using an affinity column made with an antibody to the cornified envelope precursor sciellin. Using reverse transcriptase polymerase chain reaction technology and total RNA isolated from bovine tongue epithelium, a cDNA was obtained whose nucleotide sequence coded for a protein homologous to human TLS. Nuclear staining in all layers of human epidermis and bovine stratified epithelium was observed with an antibody to TLS, whereas peripheral staining of the upper layers of these tissues was observed with the antibody to sciellin. Cultured cells gave similar results; however, adult tissue required boiling in citrate buffer to unmask antigenic sites before reacting with the TLS antibody. Western blots of extracts of human and bovine keratinocytes using TLS and sciellin antibodies showed that the two proteins shared at least one epitope, but that they were different. TLS was lost from the nucleus following inhibition of RNA POL II activity and the protein was identified in CNBr extracts of purified keratinocytes cornified envelopes by western blot. These results clearly indicate that TLS functions as an RNA binding protein in keratinocytes in vivo and in vitro. Furthermore the sequestration of TLS to the cell envelope may play a role in regulating its nuclear-cytoplasmic transport and effect its role in transcription.

In-vivo activity and pharmacodynamics of cefotaxime in combination with vancomycin in fibrin clots infected with highly penicillin-resistant Streptococcus pneumoniae.

We studied the antipneumococcal efficacy of cefotaxime and vancomycin alone and a combination of cefotaxime with various dosages of vancomycin in the treatment of prolonged (48 h) experimental fibrin clot infections in rabbits. A clinical pneumococcal strain for which MICs were 2, 0.5 and 0.5 mg/L of penicillin, cefotaxime and vancomycin respectively, was used in this study. Cefotaxime was given iv at a fixed dose of 50 mg/kg and vancomycin iv at 1, 2.5, 5, 10 or 20 mg/kg. Maximal concentrations in clots were (mean +/- S.D.): 2.1 +/- 0.9, 1.1 +/- 0.4, 1.9 +/- 1, 2.3 +/- 1.5, 3.6 +/- 0.4 and 4 +/- 0.3 mg/g, respectively. The mean half-lives of elimination from clots were 2.2 h for cefotaxime and 7 h for vancomycin. We observed the highest bacterial reductions for the highest doses of vancomycin with or without cefotaxime. The combination of intermediate doses of vancomycin with cefotaxime led to higher antibacterial effects than either monotherapy. The low dose of vancomycin gave no significant additional effect compared with cefotaxime alone. The times of regrowth were similar for cefotaxime and cefotaxime-vancomycin 1, and also for vancomycin 10 and vancomycin 20 with or without cefotaxime but were significantly delayed for the combination cefotaxime-vancomycin 2.5 and cefotaxime-vancomycin 5 as compared with vancomycin 2.5 and vancomycin 5. By using a multivariate analysis, we demonstrated that the most important parameters were Cmax (r = 0.43) and AUC (r = 0.58) for cefotaxime alone and Cmax (r = 0.70) for vancomycin alone; none of the tested parameters was found to be significantly correlated with the efficacy of the combinations of cefotaxime and vancomycin. From these findings, and under the experimental conditions used (i.e., relatively low concentrations of cefotaxime), we demonstrated that the in-vivo antibacterial efficacy of the combination of cefotaxime and vancomycin was higher than each monotherapy when the local concentrations of vancomycin were at least 1.9 mg/L.