Development of interstitial cells of Cajal in a full-term infant without an enteric nervous system

The relationship between the development of the enteric nervous system and interstitial cells of Cajal (ICC) in the human small intestine was investigated in a full-term infant who presented with intestinal pseudo-obstruction. Immunohistochemistry revealed absence of enteric nerves and ganglia but abundant c-Kit immunoreactivity associated with Auerbach's plexus (ICC-AP). However, c-Kit immunoreactivity associated with the deep muscular plexus (ICC-DMP) and intermuscular ICC was absent. Electron microscopy showed ICC-AP with a normal ultrastructure; ICC-DMP were seen but were severely injured, suggesting degeneration. In vitro recording of intestinal muscle showed slow wave activity as well as response to cholinergic stimulation. Fluoroscopic examination of the small bowel showed a variety of motor patterns, including rhythmic, propagating contractions. In conclusion, total absence of enteric nerves was associated with absence of normal ICC-DMP. However, a normal musculature, including a network of ICC-AP, allowed for generation of rhythmic, propagating contractile activity, suggesting the presence of functional motor activity.     

Septal interstitial cells of Cajal conduct pacemaker activity to excite muscle bundles in human jejunum

BACKGROUND & AIMS: Like the heart, intestinal smooth muscles exhibit electrical rhythmicity, which originates in pacemaker cells surrounding the myenteric plexus, called interstitial cells of Cajal (ICC-MY). In large mammals, ICC also line septa (ICC-SEP) between circular muscle (CM) bundles, suggesting they might be necessary for activating muscle bundles. It is important to determine their functional significance, because a loss of ICC in humans is associated with disordered motility. Our aims were therefore to determine the role of ICC-SEP in activating the thick CM in the human jejunum. METHODS: The mucosa and submucosa were removed and muscle strips were cut and pinned in cross-section so that the ICC-MY and ICC-SEP networks and the CM could be readily visualized. The ICC networks and CM were loaded with the Ca(2+) indicator fluo-4, and pacemaker and muscle activity was recorded at 36.5 +/- 0.5( degrees )C. RESULTS: Ca(2+) imaging revealed that pacemaker activity in human ICC-MY can entrain ICC-SEP to excite CM bundles. Unlike the heart, pacemaker activity in ICC-MY varied in amplitude, propagation distance, and direction, leading to a sporadic activation of ICC-SEP. CONCLUSIONS: ICC-SEP form a crucial conduction pathway for spreading excitation deep into muscle bundles of the human jejunum, necessary for motor patterns underlying mixing. A loss of these cells could severely affect motor activity.     

Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype.

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS: Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS: When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS: These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.     

The mechanism and spread of pacemaker activity through myenteric interstitial cells of Cajal in human small intestine

BACKGROUND & AIMS: It has been generally assumed that interstitial cells of Cajal (ICC) in the human gastrointestinal tract have similar functions to those in rodents, but no direct experimental evidence exists to date for this assumption. This is an important question because pathologists have noted decreased numbers of ICC in patients with a variety of motility disorders, and some have speculated that loss of ICC could be responsible for motor dysfunction. Our aims were to determine whether myenteric ICC (ICC-MY) in human jejunum are pacemaker cells and whether these cells actively propagate pacemaker activity. METHODS: The mucosa and submucosa were removed, and strips of longitudinal muscle were peeled away to reveal the ICC-MY network. ICC networks were loaded with the Ca(2+) indicator fluo-4, and pacemaker activity was recorded via high-speed video imaging at 36.5 degrees C +/- 0.5 degrees C. RESULTS: Rhythmic, biphasic Ca(2+) transients (6.03 +/- 0.33 cycles/min) occurred in Kit-positive ICC-MY. These consisted of a rapidly propagating upstroke phase that initiated a sustained plateau phase, which was associated with Ca(2+) spikes in neighboring smooth muscle. Pacemaker activity was dependent on inositol 1,4,5-triphosphate receptor-operated stores and mitochondrial function. The upstroke phase of Ca(2+) transients in ICC-MY appeared to result from Ca(2+) influx through dihydropyridine-resistant Ca(2+) channels, whereas the plateau phase was attributed to Ca(2+) release from inositol 1,4,5-triphosphate receptor-operated Ca(2+) stores. CONCLUSIONS: Each ICC-MY in human jejunum generates spontaneous pacemaker activity that actively propagates through the ICC network. Loss of these cells could severely disrupt the normal function of the human small intestine.     

Intestinal Auerbach plexus structures with NADH histochemistry in three strains of spontaneous diabetic rats

The enteric nervous system comprises two major systems: the submucosal and the myenteric plexus. The aim of this study was to describe the myenteric plexus from three strains of spontaneous diabetic rats from the histological point of view. Samples of small intestine and of proximal and distal colon were obtained fom three spontaneous diabetic rats i.e., eSS, eSMT, beta strains and 1-year old Wistar rats. Specimens were stained with NADH (beta-nicotinamide adenine dinucleotide, reduced form) histochemical technique and examined with light microscope. Microscopically little modifications in mesh-like structure of intestinal Auerbach's plexus from eSS were detected in comparison with Wistar rats samples. Intestinal plexus of eSMT and beta rats showed disruption of mesh-like structures, modifications in the slightly colored background (smooth muscle) and augmented vascularization. Small intestine and colon are affected. In short: In our spontaneously diabetic rat models, mesh-like structure of Auerbach's plexus is strain dependent.     

Characteristic motor activity of the gastrointestinal tract in fasted conscious dogs measured by implanted force transducers

Gastrointestinal contractile activity from the gastric body to the terminal ileum in conscious dogs was continuously recorded for several weeks by means of chronically implanted strain-gage force transducers. It was found that the 24-hr changes in the gastrointestinal contractile activity consisted of the two different major patterns, the digestive and interdigestive patterns. In the interdigestive state, a cyclic, recurring, caudad-moving band of strong contractions starting in the stomach and the duodenum and traversing the entire length of the small intestine was observed. When one band of strong contractions reached the distal ileum, another developed in the stomach and the duodenum again and propagated in a caudad direction. Such recycling episodes interrupted by long-lasting quiescence repeatedly occurred until the next meal. These characteristic contractile activities observed in our dogs are identical with the interdigestive myoelectric complex of the stomach and the small bowel recently reported by Code and Marlett (5). Four sequential phases (I–IV) of the migrating myoelectric complex, defined in terms of action potential activity, seem to correspond to the resting (quiescence), preceding irregular contractions, strong contractions, and subsiding contractions observed in the present study, respectively. Function and control mechanism of the interdigestive contractile activity were discussed.     

Novel human and mouse genes encoding a shank-interacting protein and its upregulation in gastric fundus of W/WV mouse

BACKGROUND AND AIMS: A division of labor exists between different classes of interstitial cells of Cajal (ICC) in the gastrointestinal tract. In the stomach and small intestine, ICC at the level of the myenteric plexus (IC-MY) act as slow wave pacemaker cells, whereas intramuscular ICC (IC-IM) in the stomach act as intermediaries in enteric motor neurotransmission. The muscle layers of the gastric fundus do not have IC-MY, therefore electric slow waves are not generated. Intramuscular ICC are absent in the gastric fundus of W/WV mutant mice, and excitatory and inhibitory motor nerve responses are reduced in these tissues. The absence of IC-IM in W/WV mutants in the fundus provides a unique opportunity to study the molecular changes that are associated with the loss of these cells. METHODS: The tissue gene expression of wild-type and W/WV mice from gastric fundus was assayed using a murine microarray chip analysis displaying a total of 8734 elements. RESULTS: Twenty-one queries were differentially expressed in wild-type and W/WV mice. One candidate gene, encoding a novel protein homologous to rat Shank-interacting protein (Sharpin) was significantly upregulated in fed and starved W/WV mice. The full-length clone of the murine gene and its human counterpart were isolated and designated as Shank-interacting protein-like 1 (SIPL1). Human SIPL1 complementary DNA encodes a protein of 345 amino acids. This gene was localized to chromosome 8. SIPL1 was abundantly expressed in human stomach and small intestine, and scarcely expressed in cecum and rectum. CONCLUSIONS: Gene analysis showed that SIPL1 differentially express in the gastric fundus of normal and W/WV mice. The upregulation of SIPL1 in the fundus of W/WV mice, and expression in the upper gastrointestinal tract suggest that the SIPL1 gene could be associated with ICC function in mice and humans.     

Plasticity of electrical pacemaking by interstitial cells of Cajal and gastric dysrhythmias in W/W mutant mice

BACKGROUND & AIMS: Interstitial cells of Cajal (ICC) generate and propagate slow waves in the stomach. Gastric peristalsis depends on a proximal-to-distal gradient in slow wave frequency. We tested whether the gastric frequency gradient was an intrinsic property of ICC and whether dysrhythmias result from disruptions of ICC networks. METHODS: We studied wild-type (WT) and W/W(V) mice, which have only myenteric (pacemaker) ICC in the stomach. ICC distributions were analyzed by Kit immunofluorescence. Pacemaking in tissues was studied by intracellular electrophysiologic recording and in cultured ICC by monitoring mitochondrial [Ca(2+)] oscillations with rhod-2 fluorescence or membrane potential with DiBAC(4)(3) fluorescence. RESULTS: Slow wave frequencies were constant throughout WT gastric muscle sheets containing corpus and antrum. Separating the antrum from the corpus caused a significant drop in antral slow wave frequency. ICC from WT antrums also displayed significantly slower pacemaker frequencies than corpus ICC, but the corpus pacemaker frequency dominated in cocultures of corpus and antrum ICC. Myenteric ICC networks were reduced in W/W(V) mice, particularly in the corpus. In W/W(V) mice, separating the antrum from the corpus failed to reduce antral slow wave frequency. Antral pacemaker frequency in ICC from W/W(V) stomachs was the same as in corpus ICC. CONCLUSIONS: The proximal-to-distal slow wave frequency gradient and entrainment of distal electrical activity by proximal pacemakers are fundamental properties of gastric ICC. Chronic depletion of ICC networks disrupts the proximal-to-distal frequency gradient, and emergence of ectopic pacemakers in the antrum may be caused by "reprogramming" of the ICC pacemaker apparatus.     

Decreased interstitial cells of Cajal in the sigmoid colon of patients with slow transit constipation

Background and aims Slow transit constipation (STC) is a colonic motor disorder that is characterized by measurably delayed movement of materials through the colon. Although abnormalities in the neuronal networks of the colon have been demonstrated in patients with STC, the etiology of STC remains unclear. Interstitial cells of Cajal (ICC) have been shown to be the pacemaker cells of the intestine and have been implied in the pathogenesis of a number of gastrointestinal motility dysfunctions, including idiopathic STC. This study aimed to determine the normal distribution of ICC within the colon of the Chinese and also to determine if ICC are decreased in Chinese STC patients.Patients and methods Twelve patients with STC and eight age-matched normal controls were studied. Specimens of sigmoid colon were obtained immediately after resection. ICC were identified with a monoclonal antibody to c-kit by an indirect immunofluorescence method. Immunostained tissues were examined with a laser scanning confocal microscope and the area occupied by ICC was calculated with an image analysis system.Results ICC were located in the external muscle layers including myenteric plexus (MP) and submucosal border (SMB). Two types of Kit-positive ICC were observed: bipolar cells characterized by one or two long processes and multipolar cells characterized by long stellate processes extending in various directions. A higher percentage of ICC was present in the MP regions and circular muscle (CM) layers compared with the SMB and longitudinal muscle (LM) layers. Tissues from STC patients showed a considerable decrease in the number of ICC located in the four regions (ICC-LM, ICC-MP, ICC-CM, ICC-SMB), especially the ICC-SMB, in which ICC almost completely disappeared.Conclusions Similar distribution of ICC was observed in the normal sigmoid colon of the Chinese. Decreased area of c-kit+ ICC may play an important role in the pathophysiology of STC. It remains to be determined whether the loss of ICC is primary or secondary to another lesion.     

High-conductance chloride channels generate pacemaker currents in interstitial cells of Cajal

BACKGROUND & AIMS: Interstitial cells of Cajal (ICCs) are responsible for slow, wave-driven, rhythmic, peristaltic motor patterns in the gastrointestinal tract. The aim was to identify and characterize the ion channels that generate the underlying pacemaker activity. METHODS: Single ion channel recordings were obtained from nonenzymatically isolated ICCs and studied by using the cell attached and inside-out configurations of the patch clamp technique. RESULTS: A high-conductance chloride channel was observed in ICCs that was spontaneously and rhythmically active at the same frequency as the rhythmic inward currents defining ICC pacemaker activity, 20-30 cycles/min at room temperature. Main conductance levels occurred between 122-144 pS and between 185-216 pS. Periodicity in the channel opening coincided with periodicity in membrane potential change, hence, at the single channel level, chloride channels were seen to be associated with the generation of rhythmic changes in membrane potential. CONCLUSIONS: ICCs harbor high-conductance chloride channels that participate in the generation of pacemaker activity and may become a target for pharmacologic treatment of gut motor disorders.     

Disruption of interstitial cells of Cajal networks after massive small bowel resection

AIM: To investigate the disruptions of interstitial cells of Cajal (ICC) in the remaining bowel in rats after massive small bowel resection (mSBR). METHODS: Thirty male Sprague-Dawley rats fitting entry criteria were divided randomly into three experimental groups (n = 10 each): Group A rats underwent bowel transection and re-anastomosis (sham) and tissue samples were harvested at day 7 post-surgery. Group B and C rats underwent 80% small bowel resection with tissue harvested from Group B rats at day 7 post-surgery, and from Group C rats at day 14 postsurgery. The distribution of ICC at the site of the resid-ual small bowel was evaluated by immunohistochemical analysis of small intestine samples. The ultrastructural changes of ICC in the remnant ileum of model rats 7 and 14 d after mSBR were analyzed by transmission electron microscopy. Intracellular recordings of slow wave oscillations were used to evaluate electrical pacemaking. The protein expression of c-kit, ICC phenotypic markers, and membrane-bound stem cell factor (mSCF) in intestinal smooth muscle of each group were detected by Western blotting. RESULTS: After mSBR, immunohistochemical analysis indicated that the number of c-kit-positive cells was dramatically decreased in Group B rats compared with sham tissues. Significant ultrastructural changes in ICC with associated smooth muscle hypertrophy were also observed. Disordered spontaneous rhythmic contractions with reduced amplitude (8.5 ± 1.4 mV vs 24.8 ± 1.3 mV, P = 0.037) and increased slow wave frequency (39.5 ± 2.1 cycles/min vs 33.0 ± 1.3 cycles/min, P = 0.044) were found in the residual intestinal smooth muscle 7 d post mSBR. The contractile function and electrical activity of intestinal circular smooth muscle returned to normal levels at 14 d post mSBR (amplitude, 14.9 ± 1.6 mV vs 24.8 ± 1.3 mV; frequency, 30.7 ± 1.7 cycles/min vs 33.0 ± 1.3 cycles/min). The expression of Mscf and c-kit protein was decreased at 7 d (P = 0.026), but gradually returned to normal levels at 14 d. The ICC and     

Achalasia: new thoughts on an old disease.

Achalasia is the most recognized esophageal motor disorder first described 300 years ago by Sir Thomas Willis. Although the cause of achalasia remains unknown, the disease is thought to occur as a result of degeneration of Auerbach's plexus in the intramural nervous network. A review of the literature reveals that there is reversibility of this disease in some patients after treatment, raising intriguing questions as to the pathogenesis of this disorder and underscoring the need for further study.     

Intermittent rectal motor activity: a rectal motor complex?

Prolonged nocturnal recordings from multiple sites in the anorectum and duodenum were performed in 12 healthy volunteers to investigate the presence and determine the characteristics of nocturnal rectal motor activity and assess any synchronicity with phase III of the small intestinal migrating motor complex. Runs of phasic contractions of two contractile frequencies (3 or 6/minute), sustained for more than three minutes, and preceded and followed by motor quiescence were observed in 10 of the 12 subjects. This phenomenon is similar to that described by other investigators and termed the 'rectal motor complex'. The runs of contractions showed considerable inter- and intrasubject variation, with a duration of 3-30 minutes (median 9.0), amplitude of 10-55 mm Hg (median 20.0), and periods of 10-420 minutes (median 55.5) between contractile activity. There was no propagation through the rectum, the phasic motor activity rarely occurred simultaneously at more than one rectal recording site. No consistent relation with phase III of the small intestinal migrating motor complex was observed in any subject. In view of these findings, we question whether this intermittent motor activity merits the term 'rectal motor complex'.     

Role of the duodenum in the control of canine gastrointestinal motility

This study was designed to determine the role of the duodenum in controlling the interdigestive migrating motor complex of the canine stomach and small intestine. The motility patterns of 4 dogs were studied before and after resection of the entire duodenum with reimplantation of bile and pancreatic ducts. Before duodenectomy, plasma concentrations of motilin and pancreatic polypeptide varied cyclically with the migrating motor complex. After duodenectomy, the migrating motor complex was abolished in the stomach in 3 of the 4 dogs during fasting. The other dog demonstrated what appeared to be an intermittent cyclic increase in gastric contractile activity, but with markedly abnormal characteristics and at irregular intervals. The jejunal migrating motor complex continued to cycle after duodenectomy in all 4 dogs, but the mean period was shorter than before duodenectomy (83 min vs. 147 min, p less than 0.01). Duodenectomy abolished the cyclic variations of plasma motilin and pancreatic polypeptide and lowered the concentration of both peptides during all phases. This study suggests that the duodenum plays an important role in the initiation of the gastric migrating motor complex and in the coordination of interdigestive gastrointestinal motor activity, possibly by the release of motilin.     

Interstitial cells of Cajal, the Maestro in health and disease

Interstitial cells of Cajal (ICC) are important players in the symphony of gut motility. They have a very signif icant physiological role orchestrating the normal peristaltic activity of the digestive system. They are the pacemaker cells in gastrointestinal (GI) muscles. Absence, reduction in number or altered integrity of the ICC network may have a dramatic effect on GI system motility. More understanding of ICC physiology will foster advances in physiology of gut motility which will help in a future break...     

Cyclic motor activity; migrating motor complex: 1985

Most of the gastrointestinal tract and the biliary tract have a cyclic motor activity. The electric counterpart of this motor activity is called cyclic myoelectric activity. A typical motor cycle in the LES, stomach, and small intestine is composed of a quiescent state, followed by progressively increasing amplitude and frequency of contractions culminating in a state of maximal contractile activity. The colonic motor cycle has only the quiescent and the contractile states. In the small intestine, these motor complexes migrate in an aborad direction, and in the colon in both orad and aborad directions. The mechanisms of initiation and migration of these complexes are best understood in the small intestine. Both the initiation and migration of these complexes seem to be controlled by enteric neural mechanisms. The functions of the enteric mechanisms may be modulated by the central nervous system and by circulating endogenous substances. The mechanisms of initiation of these complexes are not completely understood in the rest of the gastrointestinal tract and in the biliary tract. The physiologic function of these motor complexes that occur only after several hours of fast in the upper gastrointestinal tract of nonruminants may be to clean the digestive tract of residual food, secretions, and cellular debris. This function is aided by a coordinated secretion of enzymes, acid, and bicarbonate. In ruminants, phase III activity is associated with the distal propulsion of ingested food. The function of colonic motor complexes that are not coordinated with the cyclic motor activities of the rest of the gastrointestinal tract may be only to move contents back and forth for optimal absorption.     

Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.

BACKGROUND & AIMS: Development of interstitial cells of Cajal (ICC) requires signaling via Kit receptors. Kit is activated by stem cell factor (SCF), but the source of SCF in the bowel wall is unclear and controversy exists about whether enteric neurons express the SCF required for ICC development. METHODS: Glial cell line-derived neurotrophic factor (GDNF) knockout mice, which lack enteric neurons throughout most of the gut, were used to determine whether neurons are necessary for ICC development. ICC distributions were determined with Kit immunofluorescence, and function of ICC was determined by intracellular electrical recording. RESULTS: ICC were normally distributed throughout the gastrointestinal tracts of GDNF-/- mice. Intracellular recordings from aganglionic gastrointestinal muscles showed normal slow wave activity at birth in the stomach and small intestine. Slow waves developed normally in aganglionic segments of small bowel placed into organ culture at birth. Quantitative polymerase chain reaction showed similar expression of SCF in the muscles of animals with and without enteric neurons. Expression of SCF was demonstrated in isolated intestinal smooth muscle cells. CONCLUSIONS: These data suggest that enteric neurons are not required for the development of functional ICC. The circular smooth muscle layer, which develops before ICC, may be the source of SCF required for ICC development.     

Small intestinal physiology and pathophysiology

The small intestine, like the rest of the gastrointestinal tract, is an intelligent organ. It generates a wide variety of motor patterns to meet motility requirements in different situations. Its basic motor function after a meal is to mix the chyme with exocrine and intestinal secretions, agitate its contents to uniformly and evenly expose them to the mucosal surface, and to propel them distally at a rate that allows optimal absorption of food components, and reabsorption of bile. Most of these functions are performed by individual phasic contractions. In humans, the phasic contractions are largely disorganized in time and space. These contractions may cause mixing and agitation of luminal contents with slow distal propulsion. Occasionally, an individual contraction of large amplitude and long duration migrates over several centimeters and may rapidly propel the contents over this distance. In general, the spatial and temporal relationships of individual phasic contractions become less organized distally, resulting in a slower propulsion rate in the distal small intestine than in the proximal small intestine. The migrating clustered contractions generated after a meal may also be propulsive, but because of their unpredictable and irregular occurrence, their precise role in postprandial propulsion is incompletely understood. Rapidly migrating contractions may occur when the electrical control activity is obliterated by pharmacologic agents or during parasitic infections. Their effects on motility are not known yet. Between meals, when digestion is complete, the small intestine generates migrating motor complexes that help keep the small intestine clean by dislodging debris from the villi and dumping them into the colon. This may prevent decay of these materials in the small intestine and limit their contribution to bacterial overgrowth. Giant migrating contractions may perform a similar function in the distal small intestine as well as return any refluxed fecal material back to the colon. However, the major role of giant migrating contractions may be, in pathologic states, associated with abdominal cramping and diarrhea. Giant migrating contractions are associated with mass movements. Vomiting is preceded by a retrograde giant contraction. This contraction rapidly empties the contents of the proximal half of small intestine into the stomach in preparation for vomitus expulsion by contraction of abdominal and diaphragmatic muscles. The three basic mechanisms of control of spatial and temporal patterns of contractions are myogenic, neural, and chemical.(ABSTRACT TRUNCATED AT 400 WORDS)     

Esophageal ganglia and smooth muscle in the elderly

Esophageal motor dysfunction is a common finding in elderly persons, but its etiology remains unknown. We examined the histology of Auerbach's plexus and esophageal smooth muscle in autopsy material from young and old subjects. There was a significant decrease in ganglion cells per square centimeter (1685±115.9) in old subjects when compared to young controls (2253±216.9) (P<0.05). Lymphocytic infiltration of Auerbach's plexus was commonly observed in both groups of patients, but heavy lymphocytic infiltration was slightly more common in elderly subjects. No difference was found in thickness of esophageal smooth muscle between young and old subjects. It is concluded that diminution of ganglion cells rather than smooth muscle atrophy may explain the frequent occurrence of esophageal motor dysfunction in elderly persons.This study was supported by a grant from the Charlton Fund, Tufts University School of Medicine, Boston, Massachusetts 02111.