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Supplementary Figures S1-S7 with captions and Supplementary Tables

Supplementary Methods

A 21-mo-old, male Siberian hamster (Phodopus sungorus) presented with left-sided facial swelling, proptosis of the left eye, and blepharospasm of the right eye. The hamster had been used only for breeding. Because of the poor prognosis, the hamster was euthanized without additional diagnostic assays or treatments. Routine gross pathologic evaluation demonstrated exophthalmos and presumptive hyphema of the left eye, bilateral facial edema, freely movable nodules within the mesentery, white foci within the liver, and a large mass effacing the cranial pole of the right kidney. On histologic evaluation, the mesenteric nodules and liver foci expressed histiocytic marker CD163 and thus were diagnosed as sites of histiocytic sarcoma, whereas the kidney mass was a well-differentiated renal cell carcinoma. The facial swelling resulted from bilateral, chronic, severe, branching thrombi in many facial veins. Additional age-related histopathologic findings were observed in other organs, including diffuse glomerulopathy, nesidioblastosis (pancreatic islet neoformation), and multiple foci of severe cartilage degeneration in the axial skeleton. To our knowledge, this report provides the first description of histiocytic sarcoma in a Siberian hamster.

Representative longitudinal muscle strips (6 × 10 mm) from distal small intestine were obtained from rats after 1, 2, and 3 mo of streptozocin-induced diabetes. The strips were stretched to their optimum lengths and subjected to electrical field stimulation (1-ms pulse duration, 30–270 mA, 10 Hz) in the presence of Krebs solution and Krebs solution plus 10−6 M atropine. Field stimulation produced atropine-sensitive and atropine-resistant contractions in all strips. After 1 mo, significant differences in the amplitudes of the atropine-sensitive contractions were found between the diabetic rats and nondiabetic controls. Insulintreated diabetic rats showed contraction responses that were intermediate in amplitude. After 2 mo, the difference between the control and diabetic groups was less evident but still significant. After 3 mo, the previously noted difference in the atropine-sensitive contractions between the diabetic and control groups had resolved. No significant differences among the three groups were noted in the amplitudes of the atropine-resistant contractions. Field stimulation delivered at pulse durations of 50 ms in the presence of neural blockade with tetrodotoxin (5 × 10−6 M) produced similar contraction amplitudes among the three groups at any respective time phase of the study. Dose-response studies of intestinal muscle after 3 mo of untreated diabetes showed normal tension development to both bethanechol chloride and physostigmine. These results indicate that streptozocin-induced diabetes is acutely associated with defective cholinergic neuromuscular transmission in the myenteric plexus of the distal small intestine The abnormality is less evident after 2 mo of untreated diabetes and resolves spontaneously after 3 mo. Insulin treatment appears to accelerate this resolution.

Chronic GVHD (cGVHD) is dependent on the ability of donor bone marrow (BM) B cells to produce pathogenic antibody (Ab) deposited in target organs, which is associated with increased germinal centers (GCs). BTK is necessary for B cells to enter GCs and ITK is necessary for T cell activation. We hypothesized that alloreactive GC B cells and T follicular helper cells require BTK or ITK and inhibition by ibrutinib (Ib) would prevent cGVHD. We evaluated the therapeutic effect of Ib in two models of cGVHD: a MHC-disparate (B6→B10.BR) multi-organ model complicated by bronchiolitis obliterans (BO) and a sclerodermatous (SCL) minor-mismatch (LP/J→B6) model. Mice treated with Ib had a decrease in pathogenic Ab, collagen deposition and GC B cells in target organs similar to healthy control mice. In contrast to cGVHD mice, pulmonary function in Ib treated mice was similar to control mice. To confirm that BTK and ITK are required for cGVHD we used donor mice with an ablation of BTK or ITK. Absence of BTK in B cells impeded GCs, Ab deposition and BO. Mice transplanted with T cells lacking ITK and WT BM did not develop BO. In the SCL model, mice treated with Ib did not develop clinical signs of cGVHD including less alopecia and SCL lesions. Furthermore, there was a decrease in lymphocyte infiltration surrounding the bronchioles and nephrons in treated mice. The inhibition of TEC-family kinases with Ib is a novel therapeutic for multi-organ system cGVHD with BO or SCL manifestations.

Proximal and distal rat small intestine was cut into strips measuring 6.0 X 10.0 mm. Strips cut along the oral-caudal axis were called longitudinal strips, whereas those cut 90 degrees to that axis were called circular strips. Stress in circular and longitudinal muscle strips was measured continuously as they were superfused with acetylcholine, carbamylcholine, methacholine, bethanechol, or physostigmine. Resting stress during stretch, acetylcholine-stimulated active stress, and total stress were determined. Proximal circular muscle was five times as sensitive to acetylcholine as distal circular muscle (p less than 0.05); proximal longitudinal muscle was 2.8 times as sensitive to bethanechol as distal muscle (p less than 0.05). Resting, active, and total stress were similar in proximal and distal muscle, but circular muscle showed nearly twice the resting stress of longitudinal muscle at either proximal or distal sites (p less than 0.05). Physostigmine (10(-6) M) increased acetylcholine-stimulated active stress in proximal and distal circular muscle by 29% and 70%, respectively (p less than 0.05), but not in longitudinal muscle (p greater than 0.05). This difference between proximal and distal circular muscle (41%) was also significant (p less than 0.05). Thus, the proximal and distal muscle of the rat small intestine differs in its sensitivity to various cholinergic agonists, but not in its length-stress properties.

Proximal and distal rat small intestine from control, diabetic, and insulin-treated diabetic rats was cut into strips measuring 6.0 × 10.0 mm. Strips cut along the oral-caudal axis were called longitudinal strips, while those cut 90 ° to that axis were called circular strips. The strips were stretched to their optimum lengths and subjected to electrical field stimulation (0.1–1.0-ms pulse duration, 30–270 mA, 1–26 Hz) in the presence of Krebs' solution and Krebs' solution plus 10 −6 M atropine. Field stimulation produced atropine-sensitive and atropineresistant contractions in all strips. Significant differences among the three groups were found in the amplitudes of atropine-sensitive contractions in strips from distal longitudinal muscle. Controls showed the highest amplitude contractions and diabetics the lowest, whereas the insulin-treated diabetics showed contractions intermediate in amplitude. No significant differences were noted among the atropine-resistant contractions. Field stimulation delivered at pulse durations of 5.0 and 50.0 ms in the presence of neural blockade with tetrodotoxin (5 × 10 −6 M) produced similar contraction amplitudes among the three groups. These results suggest that streptozotocin-induced diabetes mellitus is associated with defective cholinergic neuromuscular transmission in the myenteric plexus of the distal small intestine. Insulin therapy seems to improve the abnormality.