Figure 1.
(A) FLAIR image from September 2011 showing characteristic lesions in a parasagittal plane. (B–F) MRI from May 2012 revealing characteristic lesions on FLAIR imaging as before (B), T1 hypointensities (C), characteristic contrast enhancing lesions post IV Gadolinium (D, E), and characteristic lesions on T2 weighted axial image (F).
Figure 2.
C. perfringens type B in a woman with RRMS, and the prevalence of C. perfringens type A in MS and healthy controls.
(A) Left panel shows PCR based genotyping of ATCC 3626 type B strain and from patient 73F. PCR products for α, β, and ε toxin are identified in both. (B) To exclude the possibility that the type B strain identified in the stool of patient 73F was a contaminant, the profile of lysogenic prophage genes was determined in the laboratory strain and in the patient isolate (right panel). ATCC 3626 reference strain possesses all three prophage insertions, whereas the patient’s strain possesses only the A6 (weakly) and N7 prophage insertions. Phage genes and PCR product size: B1RBB5, 1000 bps; B1RAA6, 300 bps; Q8SBN7, 300 bps.
Figure 3.
Isolation and Characterization of a patient-derived C. perfringens type B strain.
(A) Schematic of the isolation procedure described in methods section. Briefly, the patient’s fecal sample was resuspended in RPM, incubated overnight at 37C, and inoculated into TSC sandwich plates. Colonies with characteristic C. perfringens morphology (black colonies) were subcultured into RPM and then streaked onto Schaedler blood agar plates until pure colonies with characteristic C. perfringens morphology that exhibited beta hemolysis were isolated. Strain genotype was confirmed by PCR analysis for toxin genes. (B) Typical black C. perfringens like morphology in TSC agar from patient 73F diluted fecal RPM sample. (C) Typical C. perfringens like morphology on Schaedler blood agar plates exhibiting beta hemolysis from a subcultured TSC colony from patient 73F. (D) Cell growth of laboratory B strain (Lab B Strain) compared to the patient-derived B strain (Patient B Strain) at 47 C measured by optical density at 600 nm (OD 600) at the given time points.
Figure 4.
The prevalence of C. perfringens type A, a human commensal, was determined in MS patients and healthy controls.
Culture of stool in C. perfringens compatible growth medium revealed that 52% of health controls harbor C. perfringens type A in the gastrointestinal tract, whereas only 23% of people with MS harbor type A.
Figure 5.
Immunoreactivity to ETX in people with MS, SLE and healthy controls.
Left panel shows Western blots. The two MS blots shown are characteristic for true positives: immunoreactivity to the C. perfringens proETX protein at 37 kD but not to the other toxins present on the blot including PA63 at 63 kD. The two blots probed with SLE sera are characteristic of false positives in that immunoreactivity is also present for PA63. Controls shown are true negatives with no immunoreactivity to any of proteins present on the blot. Note that the proETX gene encodes a protein with a predicted MW of 33 kD, which runs on SDS-PAGE with an apparent MW of 37 kD. The right panel shows prevalence of immunoreactivity to ETX in serum and/or CSF of people with MS and healthy controls.
Figure 6.
ETX binds specifically to retinal vessels and myelin.
A) Frozen sections of adult mouse retina were stained with pan-vessel marker FITC labeled BSL1 (green) and Alexa 594 labeled ETX (red). A merge and enlargement of the white boxes show co-labeling of a retinal vessel. B) Fixed frozen coronal sections from adult mouse brain through the corpus callosum were stained for proteolipid protein (PLP, green), and Alexa 594-ETX (red). Intense staining with ETX is observed in all PLP-positive white matter tracts. Merged PLP and ETX images reveal essentially complete overlapping fluorescent signal. Bar = 500 µm.