Subcutaneous and transcutaneous monitoring of murine hindlimb ischemia by in vivo Raman spectroscopy.

We have investigated the development of murine hindlimb ischemia from day 1 to day 55 after femoral artery ligation (FAL) using blood flow analysis, functional tests, histopathological staining, and in vivo Raman spectroscopy. FAL resulted in hindlimb blood deprivation and the loss of functionality as attested by the blood flow analysis and functional tests, respectively. The limbs recovered a normal circulation progressively without recovering complete functionality. Histological analysis showed changes in the morphology of muscle fibers with intense inflammation. From day 22 to day 55 post-ischemia, regeneration of the myofibers was observed. Raman spectroscopic results related to subcutaneous analysis made the identification of modification in the biochemical constituents of hindlimb muscles possible during disease progression. Ischemia was characterized by a quantitative increase in the lipid content and a decrease in the protein content. The lipid to protein ratio can be used as a spectroscopic marker to score the severity of ischemia. Multivariate statistical analysis PC-LDA (Principal Component-Linear Discriminant Analysis) was used to classify all the data measured for the normal and ischemic tissues. This classification illustrated an excellent separation between the control and ischemic tissues at any time during the course of ischemic development. In vivo Raman spectroscopy was then applied to assess the potential of this technique as a screening tool to explore an ischemic disease non-invasively (transcutaneously). For this purpose, the influence of skin on the diagnostic accuracy was evaluated; transcutaneous analysis revealed the accuracy of this technique, indicating its potential in the in situ monitoring of muscle structural changes during ischemia.

Rituximab inhibits B-cell receptor signaling.

Rituximab (RTX), a monoclonal antibody directed against the CD20 protein, is a drug commonly used in the treatment of B-cell-derived lymphoid neoplasias and of antibody-mediated autoimmune diseases. In addition to cell- and complement-mediated B-cell depletion, RTX is thought to inhibit B-cell survival and proliferation through negative regulation of canonical signaling pathways involving Akt, ERK, and mammalian target of rapamycin. However, surprisingly, although B-cell receptor (BCR) signaling has been considered critical for normal and more recently, for neoplastic B cells, the hypothesis that RTX could target BCR has never been investigated. Using follicular lymphoma cell lines as models, as well as normal B cells, we show here, for the first time, that pretreatment with RTX results in a time-dependent inhibition of the BCR-signaling cascade involving Lyn, Syk, PLC gamma 2, Akt, and ERK, and calcium mobilization. The inhibitory effect of RTX correlates with decrease of raft-associated cholesterol, complete inhibition of BCR relocalization into lipid raft microdomains, and down-regulation of BCR immunoglobulin expression. Thus, RTX-mediated alteration of BCR expression, dynamics, and signaling might contribute to the immunosuppressive activity of the drug.