Muscle biopsy substantiates long-term MRI alterations one year after a single dose of botulinum toxin injected into the lateral gastrocnemius muscle of healthy volunteers.

Despite numerous clinical and experimental studies on botulinum toxin type A (BoNT/A), long-term alterations of muscle texture and fine structure following BoNT/A treatment have thus far not been studied in normal human skeletal muscle. After obtaining institutional review board approval, we performed a prospective, placebo-controlled, double-blinded follow-up study on two healthy adults using magnetic resonance imaging (MRI) and muscle biopsy to visualize long-term alterations after a single BoNT/A injection into the lateral head of the gastrocnemius muscle. MRI disclosed a high-signal-intensity pattern in short tau inversion recovery sequences, and a reduction of the cross-sectional area in the BoNT/A-injected, but not in the saline-injected contralateral control muscle (at 6 to 9 months in volunteer A: 73%, in B: 62%; at 12 months in A: 88%, and in B: 78%). Enzyme histochemistry, 12 months after injection, confirmed neurogenic atrophy of muscle fibers only in the BoNT/A-injected muscle. Electron microscopy revealed additional degenerative changes at the neuromuscular junction. The data confirm that MRI is a suitable tool to monitor the long-term effect of BoNT/A on skeletal muscle. Neurogenic muscle atrophy following a single BoNT/A injection should be taken into consideration when repeated BoNT/A injections into the same muscles are proposed.

Automated classification of breast parenchymal density: topologic analysis of x-ray attenuation patterns depicted with digital mammography.

OBJECTIVE: We used an algorithm for quantitative image processing to classify breast tissue into the categories fibrosis, involution atrophy, and normal. The algorithm entailed use of Minkowski functionals in topologic analysis of x-ray attenuation patterns on digital mammograms. The results were compared with those of techniques based on evaluation of gray-level histograms. MATERIALS AND METHODS: One hundred digital mammograms were classified by consensus of two experienced readers. A topologic parameter extracted from the Minkowski functional spectra was obtained for retromammilar image sections (512 x 512 pixels). From the gray-level histogram of each of these samples, the 20th percentile, median, and mean were determined. Discriminant analysis was used to assess the predictive value of the methods with respect to correct categorization. RESULTS: The mean gray-level intensity of normal breast tissue was 90 +/- 9, and the 20th percentile was 68 +/- 18. The mean gray-level intensity was 84 +/- 7 for involution and 90 +/- 8 for fibrosis; the 20th percentile was 75 +/- 6 for involution and 73 +/- 10 for fibrosis. The results of discriminant analysis showed that use of the gray-level histogram parameters led to correct classification in 66% of cases. Use of topologic analysis with Minkowski functionals increased the rate of correct classification to 83%. When a combined model of histogram-derived parameters and Minkowski functionals was used, 89% of cases were categorized correctly. CONCLUSION: Topologic analysis of x-ray attenuation patterns on digital mammograms obtained with Minkowski functionals is simple and robust, and the results agree with radiologists' ratings. Because correct classification is significantly higher than with use of density features, our technique may be an objective and quantitative alternative in the evaluation of the parenchymal structure of the breast.

Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides.

The Toll-like receptor (TLR)9 is critical for the recognition of immunostimulatory CpG motifs but may cooperate with other TLRs. We analyzed TLR1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of human PBMC and determined the sensitivity of these subsets to CpG oligodeoxynucleotides (ODN). TLR1 and TLR6 were expressed in all cell types examined. TLR10 was highly expressed in B cells and weakly expressed in plasmacytoid dendritic cells (PDC). High expression of TLR2 was characteristic for monocytes. PDC and B cells expressed marked levels of TLR7 and TLR9 and were directly sensitive to CpG ODN. In CpG ODN-stimulated PDC and B cells, TLR9 expression rapidly decreased, as opposed to TLR7, which was up-regulated in PDC and decreased in B cells. In monocytes, NK cells, and T cells, TLR7 was absent. Despite low expression of TLR9, monocytes, NK cells, and T cells did not respond to CpG ODN in the absence of PDC but were activated in the presence of PDC. In conclusion, our studies provide evidence that PDC and B cells, but not monocytes, NK cells, or T cells, are primary targets of CpG ODN in peripheral blood. The characteristic expression pattern of TLR1-10 in cellular subsets of human PBMC is consistent with the concept that TLR9 is essential in the recognition of CpG ODN in PDC and B cells. In addition, selective regulation of TLR7 expression in PDC and B cells by CpG ODN revealed TLR7 as a candidate TLR potentially involved in modulating the recognition of CpG motifs.

CpG-A and CpG-B oligonucleotides differentially enhance human peptide-specific primary and memory CD8+ T-cell responses in vitro.

Two distinct types of CpG oligodeoxynucleotide (ODN) have been identified that differ in their capacity to stimulate antigen-presenting cells: CpG-A induces high amounts of interferon-alpha (IFN-alpha) and IFN-beta in plasmacytoid dendritic cells (PDCs), whereas CpG-B induces PDC maturation and is a potent activator of B cells but stimulates only small amounts of IFN-alpha and IFN-beta. Here we examined the ability of these CpG ODNs to enhance peptide-specific CD8+ T-cell responses in human peripheral blood mononuclear cells (PBMCs). The frequency of influenza matrix-specific "memory" CD8+ T cells was increased by both types of CpG ODN, whereas the frequency of Melan-A specific "naive" CD8+ T cells increased on stimulation with CpG-B but not with CpG-A. The presence of PDCs in PBMCs was required for this CpG ODN-mediated effect. The expanded cells were cytotoxic and produced IFN- on peptide restimulation. Soluble factors induced by CpG-A but not CpG-B increased the granzyme-B content and cytotoxicity of established CD8+ T-cell clones, each of which was IFN-alpha/-beta dependent. In conclusion, CpG-B seems to be superior for priming CD8+ T-cell responses, and CpG-A selectively enhances memory CD8+ T-cell responses and induces cytotoxicity. These results demonstrate distinct functional properties of CpG-A and CpG-B with regard to CD8 T cells.