Andrew M. Yeager

Pneumatosis intestinalis in children after allogeneic bone marrow transplantation

Four children, ages 3 to 8 years, developed pneumatosis intestinalis (PI) after allogeneic bone marrow transplantation (BMT) for acute leukemia or severe aplastic anemia. PI was detected at a median of 48 days (range, 10–63 days) after BMT and was associated with abdominal symptoms and clinical signs. All patients had severe systemic and/or high-grade cutaneous acute graft-versus-host disease (AGVHD) at some time after BMT and were receiving corticosteroids at the time of development of PI; however, PI was associated with concomitant severe AGVHD in only one patient. One patient with PI hadHafnia alvei bacteremia and another patient had gastroenteritis due to rotavirus and adenovirus. All patients were treated with supportive care and systemic broad-spectrum antibiotics, and PI resolved 2–16 days after onset. Two patients died with BMT-associated complications unrelated to PI. Multiple factors contribute to the development of PI after BMT, and the prognosis for recovery from PI is good with medical management alone. Overall survival in these patients is dependent on the frequency and severity of other conditions, such as AGVHD and opportunistic infections, after BMT.

Nerve conduction studies in the Twitcher mouse (murine globoid cell leukodystrophy)

Progression of the neuropathy in the Twitcher mouse (twi-C57BL/6J), an animal model of globoid cell leukodystrophy, was assessed with serial motor nerve conduction studies from just after birth until near death (day 45) and after hematopoietic cell transplantation (HCT). Under ether anesthesia, the tibial nerve was stimulated percutaneously at the sacral notch and at the ankle, and recordings were made from plantar foot muscles. Motor conduction velocity (MCV), distal latency, amplitude, duration and number of phases of compound muscle action potentials on proximal (pCMAP) and distal (dCMAP) stimulation were measured. In 15-19 day-old Twitcher, despite the absence of motor signs, MCV was significantly reduced, 12.8 +/- 2.8 (10) m/s (M +/- SD, No. of recordings), compared with unaffected siblings, 18.1 +/- 2.6 (21) m/s (P less than 0.01). The ratio of pCMAP to dCMAP amplitudes was reduced in the Twitcher, 0.39 +/- 0.13 (10), compared with controls 0.72 +/- 0.17 (21) and the ratio of pCMAP to dCMAP phases was increased (2.8 +/- 0.8 (10) vs 1.0 +/- 0.2 (21), P less than 0.01 for all). As neurologic signs progressed by 35-39 days, MCV became slower, 5.8 +/- 1.0 (11) m/s, pCMAP and dCMAP became smaller, but the ratio of pCMAP to dCMAP amplitudes in the Twitcher (0.55 +/- 0.36, 11) was similar to controls (0.71 +/- 1.0, 20) as was the ratio of pCMAP to dCMAP phases (1.0 +/- 0.4 vs 1.0 +/- 0.1). These results suggest that there is diffuse non-uniform slowing of nerve conduction with block especially in proximal nerve fibers initially. With HCT, mean MCV remained slow (6.7 +/- 1.2 (18) m/s, vs 34.5 +/- 3.9 (12) m/s) but motor function persisted.

Simultaneous determination of psychosine and cerebrosides.

A new method was developed for the simultaneous determination of psychosine and cerebrosides in tissues. Total lipids extracted from the tissues were treated with [3H]acetic anhydride in toluene-methanol. Known amounts of nonradioactive N-acetylpsychosine were added to the reaction product and then subjected to mild alkaline methanolysis. After the product was washed, it was fractionated by silica gel column chromatography and the fraction containing glycolipids was benzoylated. The benzoylated product was finally fractionated on TLC. The amounts of benzoylated derivatives of nonhydroxy- and hydroxycerebrosides and N-acetylpsychosine were determined using a scanning densitometer. The amounts of psychosine in tissues were calculated from the radioactivity in the spot of N-acetylpsychosine and the recovery of added carrier N-acetylpsychosine. This method allowed us to determine 5 to 1000 pmol of psychosine and 1 to 20 nmol of cerebrosides in peripheral nerves and other tissues of the twitcher mouse as well as transfected Schwann cells derived from the sciatic nerves.

Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse

Abstract: The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide β‐galactosidase activity. Saposin A and C, the heat‐stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide β‐galactosidase as well as glucosylceramide β‐glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide β‐glucosidase (measured by hydrolysis of 4‐methylumbelliferyl‐β‐glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide 4bT‐galactosidase as well as 4‐methylum‐belliferyl‐β‐glucoside β‐glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide β‐galactosidase.

Hematopoietic Cell Transplantation in Murine Globoid Cell Leukodystrophy (The Twitcher Mouse)—A Model of a Human Sphingolipidosis (Krabbe Disease)

Human lysosomal storage diseases are associated with significant morbidity and mortality, and the management of these conditions has been limited to symptomatic supportive care. Allogeneic bone marrow transplantation (BMT) may be therapeutic in these disorders by repopulating the lymphohematopoietic and reticuloendothelial systems with enzymatically normal donor-derived cells, thus providing a stable, self-renewing source of enzyme(s) in which the recipient is deficient [1,2]. Despite several favorable preliminary clinical reports [3–10], the long-term therapeutic effects of BMT in patients with genetic storage diseases are not well known.

AMNIOTIC EPITHELIAL CELL IMPLANTATION (AECI) IN LYSOSOMAL STORAGE DISEASES

Human amniotic epithelium expresses very low levels of HLA antigens on the cell surface and is non-immunogenic when implanted subcutaneously into normal recipients (Nature 295: 325, 1982). Amniotic epithelial cells also produce several lysosomal hydrolases (Lancet 2: 1003, 1981). We performed AECI as possible enzyme replacement therapy in 5 patients, ages 22 mos-7 yrs, with heritable storage diseases. One patient had Gm1 gangliosidosis, two had Farbers disease, one had metachromatic leukodystrophy, and one had mucopolysaccharidosis I Hurler-Scheie compound. Human amnion was obtained from elective caesarean deliveries and implanted subcutaneously into recipients within 6 hr after procurement. All patients tolerated the implantation well. In the post-AECI period, serum and leukocyte samples were obtained from recipients and assayed for enzyme activity. Most patients also had quantitation of urinary substrate at selected times after AECI. All patients have been followed for 3-6 mos post implant. We have not detected any increased enzyme activity or alterations in urinary excretion of substrate after AECI, and no patient has demonstrated objective clinical improvement. In our experience, AECI is well tolerated but fails to provide a source of replacement enzyme in selected storage diseases.