Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

In aged mice, outgrowth of intraocular melanoma depends on proangiogenic M2-type macrophages.

Macrophages are part of the tumor microenvironment and have been associated with poor prognosis in uveal melanoma. We determined the presence of macrophages and their differentiation status in a murine intraocular melanoma model. Inoculation of B16F10 cells into the anterior chamber of the eye resulted in rapid tumor outgrowth. Strikingly, in aged mice, tumor progression depended on the presence of macrophages, as local depletion of these cells prevented tumor outgrowth, indicating that macrophages in old mice had a strong tumor-promoting role. Immunohistochemistry and gene expression analysis revealed that macrophages carried M2-type characteristics, as shown by CD163 and peroxisome proliferator-activated receptor gamma expression, and that multiple angiogenic genes were heavily overrepresented in tumors of old mice. The M2-type macrophages were also shown to have immunosuppressive features. We conclude that tumor-associated macrophages are directly involved in tumor outgrowth of intraocular melanoma and that macrophages in aged mice have a predisposition for an M2-type profile.

The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.